α-O-linked glycoconjugates, methods of preparation and uses thereof

ABSTRACT

The present invention provides novel α-O-linked glycoconjugates such as α-O-linked glycopeptides, as well convergent methods for synthesis thereof. The general preparative approach is exemplified by the synthesis of the mucin motif commonly found on epithelial tumor cell surfaces. The present invention further provides compositions and methods of treating cancer using the α-O-linked glycoconjugates.

This application is based on U.S. Provisional Application Serial No.60/043,713, filed April 16, 1997, the contents of which are herebyincorporated by reference into this application.

This invention was made with government support under grants CA-28824,HL-25848 and Al-16943 from the National Institutes of Health.Additionally, the present invention was supported in part by afellowship from the United States Army to Scott Kuduk (DAMD17-98-1-8154). Accordingly, the U.S. Government has certain rights inthe invention.

FIELD OF THE INVENTION

The present invention is in the field of α-O-linked glycopeptides. Inparticular, the present invention relates to methods for the preparationof α-O-linked glycoconjugates with clustered glycodomains which areuseful as anticancer therapeutics. The present invention also providesnovel compositions comprising such α-O-linked glycoconjugates andmethods for the treatment of cancer using these glycoconjugates.

Throughout this application, various publications are referred to, eachof which is hereby incorporated by reference in its entirety into thisapplication to more fully describe the state of the art to which theinvention pertains.

BACKGROUND OF THE INVENTION

The role of carbohydrates as signaling molecules in the context ofbiological processes has recently gained prominence. M. L. Phillips, etal., Science, 1990, 250, 1130; M. J. Polley, et al., Proc. Natl. Acad.Sci. USA, 1991 88, 6224: T. Taki, et al., J. Biol. Chem., 1996, 261,3075; Y. Hirabayashi, A. Hyogo, T. Nakao, K. Tsuchiya, Y. Suzuki, M.Matsumoto, K. Kon, S. Ando, ibid., 1990, 265, 8144; O. Hindsgaul, T.Norberg, J. Le Pendu, R. U. Lemieux, Carbohydr. Res. 1982, 109, 109; U.Spohr, R. U. Lemieux, ibid., 1988, 174, 211). The elucidation of thescope of carbohydrate involvement in mediating cellular interaction isan important area of inquiry in contemporary biomedical research. Thecarbohydrate molecules, carrying detailed structural information, tendto exist as glycoconjugates (cf. glycoproteins and glycolipids) ratherthan as free entities. Given the complexities often associated withisolating the conjugates in homogeneous form and the difficulties inretrieving intact carbohydrates from these naturally occurringconjugates, the applicability of synthetic approaches is apparent. (Forrecent reviews of glycosylation see: Paulsen, H.; Angew. Chemie Int. Ed.Engl. 1982, 21, 155; Schmidt, R. R., Angew. Chemie Int. Ed. Engl. 1986,25, 212; Schmidt, R. R., Comprehensive Organic Synthesis,Vol. 6, Chapter1(2), Pergamon Press, Oxford, 1991; Schmidt, R. R., Carbohydrates,Synthetic Methods and Applications in Medicinal Chemistry,Part I,Chapter 4, VCH Publishers, Weinheim, New York, 1992. For the use ofglycals as glycosyl donors in glycoside synthesis, see Lemieux, R. U.,Can. J. Chem., 1964, 42, 1417; Lemieux, R. U., Fraiser-Reid, B., Can. J.Chem. 1965, 43, 1460; Lemieux, R. U.; Morgan, A. R., Can. J. Chem. 1965,43, 2190; Thiem, J., et al., Synthesis 1978, 696; Thiem, J. Ossowski,P., Carbohydr. Chem., 1984, 3, 287; Thiem, J., et al., Liebigs Ann.Chem., 1986, 1044; Thiem, J. in Trends in Synthetic CarbohydrateChemistry, Horton, D., et al., eds., ACS Symposium Series No. 386,American Chemical Society, Washington, D.C., 1989, Chapter 8.)

The carbohydrate domains of the blood group substances contained in bothglycoproteins and glycolipids are distributed in erythrocytes,epithelial cells and various secretions. The early focus on thesesystems centered on their central role in determining blood groupspecificities. R. R. Race; R. Sanger, Blood Groups in Man, 6th ed.,Blackwell, Oxford, 1975. However, it is recognized that suchdeterminants are broadly implicated in cell adhesion and bindingphenomena. (For example, see M. L. Phillips, et al., Science 1990, 250,1130.) Moreover, ensembles related to the blood group substances inconjugated form are encountered as markers for the onset of varioustumors. K. O. Lloyd, Am. J. Clinical Path., 1987, 87, 129; K. O. Lloyd,Cancer Biol., 1991, 2, 421. Carbohydrate-based tumor antigenic factorshave applications at the diagnostic level, as resources in drug deliveryor ideally in immunotherapy. Toyokuni, T., et al., J. Am. Chem Soc.1994, 116, 395; Dranoff, G., et al., Proc. Natl. Acad. Sci. USA 1993,90, 3539; Tao, M -H.; Levy, R., Nature 1993, 362, 755; Boon, T., Int. J.Cancer 1993, 54, 177; Livingston, P. O., Curr. Opin. Immunol. 1992, 4,624; Hakomori, S., Annu. Rev. Immunol. 1984, 2, 103; K. Shigeta, et al.,J. Biol. Chem. 1987, 262, 1358.

The present invention provides new strategies and protocols forglycopeptide synthesis. The object is to simplify such preparations sothat relatively complex domains can be assembled with highstereospecifity. Major advances in glycoconjugate synthesis require theattainment of a high degree of convergence and relief from the burdensassociated with the manipulation of blocking groups. Another requirementis that of delivering the carbohydrate determinant with appropriateprovision for conjugation to carrier proteins or lipids. Bernstein, M.A.; Hall, L. D., Carbohydr. Res. 1980, 78, Cl; Lemieux, R. U., Chem.Soc. Rev. 1978, 7, 423; R. U. Lemieux, et al., J. Am. Chem. Soc. 1975,97, 4076. This is a critical condition if the synthetically derivedcarbohydrates are to be incorporated into carriers suitable for clinicalapplication.

Antigens which are selective (or ideally specific) for cancer cellscould prove useful in fostering active immunity. Hakomori, S., CancerRes., 1985, 45, 2405-2414; Feizi, T., Cancer Surveys 1985, 4, 245-269.Novel carbohydrate patterns are often presented by transformed cells aseither cell surface glycoproteins or as membrane-anchored glycolipids.In principle, well chosen synthetic glycoconjugates which stimulateantibody production could confer active immunity against cancers whichpresent equivalent structure types on their cell surfaces. Dennis, J.,Oxford Glycostems Glyconews, Second Ed., 1992; Lloyd, K. O., in SpecificImmunotherapy of Cancer with Vaccines, 1993, New York Academy ofSciences, pp.50-58. Chances for successful therapy improve withincreasing restriction of the antigen to the target cell. For example,one such specific antigen is the glycosphingolipid isolated by Hakomoriand collaborators from the breast cancer cell line MCF-7 andimmunocharacterized by monoclonal antibody MBrl. Bremer, E. G., et al.,J. Biol. Chem. 1984, 259, 14773-14777; Menard, S., et al., Cancer Res.1983, 43, 1295-1300.

The surge of interest in glycoproteins (M. J. McPherson, et al., eds.,PCR A Practical Approach, 1994, Oxford University Press, Oxford, G. M.Blackburn; M. J. Gait, Eds., Nucleic Acids in Chemistry and Biology,1990, Oxford University Press, Oxford; A. M. Bray; A. G. Jhingran; R. M.Valero; N. J. Maeji, J. Org. Chem. 1944, 59, 2197; G. Jung; A. G.Beck-Sickinger, Angew Chem. Int. Ed. Engl. 1992, 31, 367; M. A. Gallop;R. W. Barrett; W. J. Dower; S. P. A. Fodor; E. M. Gordon, J. Med. Chem.1994, 37, 1233; H. P. Nestler; P. A. Bartlett; W. C. Still, J. Org.Chem. 1994, 59, 4723; M. Meldal, Curr. Opin. Struct. Biol. 1994, 4, 673)arises from heightened awareness of their importance in diversebiochemical processes including cell growth regulation, binding ofpathogens to cells (O. P. Bahl, in Glycoconjugates: Composition,structure, and function,H. J. Allen, E. C. Kisailus, Eds., 1992, MarcelDekker, Inc., New York, p.1), intercellular communication and metastasis(A. Kobata, Acc. Chem. Res. 1993, 26, 319). Glycoproteins serve as celldifferentiation markers and assist in protein folding and transport,possibly by providing protection against proteolysis. G. Opdenakker, etal., FASEB J. 1993, 7, 1330. Improved isolation techniques andstructural elucidation methods (A. De; K. -H. Khoo, Curr. Opin. Struct.Biol. 1993, 3, 687) have revealed high levels of microheterogeneity innaturally-produced glycoproteins. R. A. Dwek, et al., Annu. Rev.Biochem. 1993, 62, 65. Single eukaryotic cell lines often produce manyglycoforms of any given protein sequence. For instance, erythropoietin(EPO), a clinically useful red blood cell stimulant against anemia, isglycosylated by more than 13 known types of oligosaccharide chains whenexpressed in Chinese hamster ovary cells (CHO) (Y. C. Lee; R. T. Lee,Eds., Neoglycoconjugates: Preparation and Applications, 1994, AcademicPress, London). The efficacy of erythropoietin is heavily dependent onthe type and extent of glycosylation (E. Watson, et al., Glycobiology,1994, 4, 227).

Elucidation of the biological relevance of particular glycoproteinoligosaccharide chains requires access to pure entities, heretoforeobtained only by isolation. Glycoprotein heterogeneity renders thisprocess particularly labor-intensive. However, particular cell lines canbe selected to produce more homogeneous glycoproteins forstructure-activity studies. U.S. Pat. No. 5,272,070. However, theproblem of isolation from natural sources remains difficult.

Receptors normally recognize only a small fraction of a givenmacromolecular glycoconjugate. Consequently, synthesis of smaller butwell-defined putative glycopeptide ligands could emerge as competitivewith isolation as a source of critical structural information (Y. C.Lee; R. T. Lee, Eds., supra).

Glycoconjugates prepared by total synthesis are known to inducemobilization of humoral responses in the murine immune system.Ragupathi, G., et al., Angew. Chem. Int. Ed. Engl. 1997, 36, 125;Toyokuni, T.; Singhal, A. K., Chem. Soc. Rev. 1995, 24, 231; Angew.Chem. Int. Ed. Engl. 1996, 35, 1381. Glycopeptides, in contrast to mostglycolipids and carbohydrates themselves, are known to bind to majorhistocompatability complex (MHC) molecules and stimulate T cells infavorable cases. Deck, B., et al., J. Immunology 1995, 1074; Haurum, J.S., et al., J. Exp. Med. 1994, 180, 739; Sieling, P. A., et al., Science1995, 269, 227 (showing T cell recogniztion of CD1-restricted microbialglycolipid). Properly stimulated T cells express receptors thatspecifically recognize the carbohydrate portion of a glycopeptide. Thepresent invention demonstrates a means of augmenting the immunogenicityof carbohydrates by use of a peptide attachment.

Preparation of chemically homogeneous glycoconjugates, includingglycopeptides and glycoproteins, constitutes a challenge of highimportance. Bill, R. M.; Flitsch, S. L.; Chem. & Biol. 1996, 3, 145.Extension of established cloning approaches to attain these goals arebeing actively pursued. Various expression systems (including bacteria,yeast and cell lines) provide approaches toward this end, but, as notedabove, produce heterogeneous glycoproteins. Jenkins, N., et al., NatureBiotech. 1996, 14, 975. Chemical synthesis thus represents a preferredavenue to such bi-domainal constructs in homogeneous form. Moreover,synthesis allows for the assembly of constructs in which selectedglycoforms are incorporated at any desired position of the peptidechain.

Prior to the subject invention, methods of glycopeptide synthesispioneered by Kunz and others allowed synthetic access to homogenoustarget systems both in solution and solid phase (M. Meldal, Curr. Opin.Struct. Biol, 1994, 4, 710; M. Meldal, in Neoglycoconjugates:Preparation and Applications, supra; S. J. Danishefsky; J. Y. Roberge,in Glycopeptides and Related Compounds: Chemical Synthesis, Analysis andApplications, 1995, D. G. Large, C. D. Warren, Eds., Marcel Dekker, NewYork; S. T. Cohen-Anisfeld and P. T. Lansbury, Jr., J. Am. Chem. Soc.,1993, 115, 10531; S. T. Anisfeld; P. T. Lansbury jr., J. Org. Chem,1990, 55, 5560; D. Vetter, et al., Angew. Chem. Int. Ed. Engl, 1995, 34,60-63). Cohen-Anisfeld and Lansbury disclosed a convergentsolution-based coupling of selected already available saccharides withpeptides. S. T. Cohen-Anisfeld; P. T. Lansbury, Jr., J. Am. Chem. Soc.,supra.

Thus, few effective methods for the preparation of αO-linkedglycoconjugates were known prior to the present invention. Nakahara, Y.,et al., In Synthetic Oligosaccharides, ACS Symp. Ser. 560, 1994, pp.249-266; Garg, H. G., et al., Adv. Carb. Chem. Biochem. 1994, 50, 277.Nearly all approaches incorporated the amino acid (serine or threonine)at the monosaccharide stage. This construction would be followed byelaboration of the peptidyl and carbohydrate domains in a piecemealfashion. Qui, D.; Koganty, R. R.; Tetrahedron Lett. 1997, 38, 45.Eloffson, M., et al., Tetrahedron 1997, 53, 369. Meinjohanns, E., etal., J. Chem. Soc., Perkin Trans. 1, 1996, 985. Wang, Z -G., et al.,Carbohydr. Res. 1996, 295, 25. Szabo, L., et al., Carbohydr. Res. 1995,274, 11. The scope of the synthetic problem is well known in the art,but little progress has been achieved. The present invention provides analternate, simpler and more convergent approach (FIG. 2).

Toyokuni et al., J. Amer. Chem. Soc., 1994, 116, 395, have preparedsynthetic vaccines comprising dimeric Tn antigen-lipopeptide conjugateshaving efficacy in eliciting an immune response against Tn-expressingglycoproteins. However, prior to investigations of the presentinventors, it was not appreciated that the surface of prostate cancercells presents glycoproteins comprising Tn clusters linked via threoninerather than serine residues. Accordingly, the present invention providesa vaccine having unexpectedly enhanced anticancer efficacy.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide novelα-O-linked glycoconjugates including glycopeptides and related compoundswhich are useful as anticancer therapeutics.

Another object of the present invention is to provide synthetic methodsfor preparing such glycoconjugates. An additional object of theinvention is to provide compositions useful in the treatment of subjectssuffering from cancer comprising any of the glycoconjugates availablethrough the preparative methods of the invention, optionally incombination with pharmaceutical carriers.

The present invention is also intended to provide a fully syntheticcarbohydrate vaccine capable of fostering active immunity in humans.

A further object of the invention is to provide methods of treatingsubjects suffering from of cancer using any of the glycoconjugatesavailable through the preparative methods of the invention, optionallyin combination with pharmaceutical carriers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic structure for α-O-linked glycoconjugates aspresent in mucins.

FIG. 2 provides a general synthetic strategy to mucin glycoconjugates.

FIG. 3 provides a synthetic route to prepare key intermediateβ-phenylthioglycoside 11. Reaction conditions: (a) (1) DMDO, CH₂Cl₂; (2)6-O-TIPS-galactal, ZnCl₂, −78° C. to 0° C. (3) Ac₂O, Et₃N, DMAP, 75%;(b) TBAF/AcOH/THF; 80%; (c) 5 (1.3 eq), TMSOTf (0.1 eq), THF:Toluene1:1, −60° C. to −45° C., 84%, α:β4:1; (d) NaN₃, CAN, CH₃CN, −15° C.,60%; (e) LiBr, CH₃CN, 75%; (f) (1)1 PhSH, iPr₂NEt, CH₃CN, 82% (2)CCl₃CN, K₂CO₃, CH₂CH₂, 80%; (g) (1) PhSH, iPr₂NEt; (2) CIP(OEt)₂,iPr₂NEt, THF, (labile compd, −72% for two steps); (h) (1) LiBr, CH₃CN,75%; (2) LiSPh, THF, 0° C., 70%).

FIG. 4 presents a synthetic route to glycoconjugate mucin 1. Reactionconditions: (a) CH₃COSH, 78%; (b) H₂/10% Pd-C, MeOH, H₂O, quant.; (c)H₂N-Ala-Val-OBn, IIDQ, CH₂Cl₂, 85%; (d) KF, DMF, 18-crown-6, 95%; (e)15, IIDQ, 87%; (f KF, DMF, 18-crown-6,93%; (g) 14, IIDQ, 90%; (h) (1)KF, DMF, 18-crown-6; (2) Ac₂O, CH₂Cl₂,: (i) H₂/10% Pd-C, MeOH, H₂O, 92%(three steps); (j) NaOH, H₂O, 80%.,

FIG. 5 shows a synthetic route to prepare glycoconjugates by a fragmentcoupling. Reagents: (a) IIDQ, CH₂Cl₂, rt, 80%; (b) H₂/Pd-C, MeOH, H₂O,95%; (c) CF₃COOH, CH₂Cl₂; (d) NaOH, H₂O, MeOH.

FIG. 6 shows the synthesis of α-O-linked glycopeptide conjugates of theLe^(y) epitope via an iodosulfonamidation/4+2 route.

FIG. 7 provides the synthesis of α-O-linked glycopeptide conjugates ofthe Le^(y) epitope via an azidonitration/4+2 route.

FIGS. 8 and 9 present examples of glycopeptides derived by the method ofthe invention.

FIG. 10 illustrates a synthetic pathway to prepare glycopeptides ST_(N)and T(TF).

FIG. 11 shows a synthetic pathway to prepare glycopeptide (2,3)ST.

FIG. 12 shows a synthetic pathway to prepare the glycopeptideglycophorine.

FIG. 13 presents a synthetic pathway to prepare glycopeptides 3-Le^(y)and 6-Le^(y).

FIG. 14 provides a synthetic pathway to prepare T-antigen.

FIG. 15 shows a synthetic pathway to prepare the alpha cluster of theT-antigen.

FIG. 16 shows a synthetic pathway to prepare the beta cluster of theT-antigen. The sequence of reactions are as represented in FIG. 15.

FIGS. 17, 18 and 19 presents a synthesis of α-O-linked glycopeptideconjugates of the Le^(y) epitope. R is defined in FIG. 18.

FIG. 20 shows (A) the conjugation of Tn-trimer glycopeptide to PamCyslipopeptide; (B) a general representation of a novel vaccine construct;and (C) a PamCys Tn Trimer.

FIG. 21 illustrates (A) a method of synthesis of a PamCys-Tn-trimer 3;and (B) a method of preparation of KLH and BSA conjugates (12, 13) viacross-linker conjugation.

FIG. 22 shows (A) a mucin related F1α antigen and a retrosyntheticapproach to its preparation; and (B) a method of preparing intermediates5′ and 6′. conditions: i) NaN₃, CAN, CH₃, CN, −20° C., overnight, 40%, α(4a′): β (4b′) 1:1; ii) PhSH, EtN(i-Pr)₂, CH₃,CN, 0° C., 1 h, 99.8%,iii) K₂CO₃, CCl₃,CN, CH₂Cl₂, rt, 5h, 84%, 5a′: 5b′ 1:5;iv) DAST, CH₂Cl₂,0 ° C., 1 h, 93%, 6a′: 6b′ 1:1.

FIG. 23 shows a method of preparing intermediates 1′ and 2′. Conditions:i) TBAF, HOAc, THF, rt, 3d, 100% yield for 9′, 94% yield for 10′; ii)11′, BF₃,Et₂O, −30° C., overnight; iii) AcSH, pyridine, rt, overnight,72% yield yield based on 50% conversion of 11′, 58% yield based on 48%conversion of 12(two steps); iv) 80% aq. HOAc, overnight, rt-40 ° C; v)Ac₂O, pyridine, rt., overnight; vi) 10% Pd/C, H₂, MeOH-H₂O, rt, 4 h;vii) Morpholine, DMF, rt, overnight; viii) NaOMe, MeOH-THF, rt,overnight, 64% yield for 1′, 72% yield for 2′ (five steps).

FIG. 24 shows a method of preparing intermediates in the synthesis ofF1α antigen. Conditions: i) (sym-collidine)₂ClO₄, PhSO₂NH₂, 0° C.;LiHMDS<EtSH, −40 ° C.-rt, 88% yield in two steps; ii) MeOTf, DTBP, 0°C., 86% yield for 20′ plus 8% yield of α isomer; 85% yield for 21′ plus6% yield of α isomer; iii) Na, NH₃, 78° C.; Ac₂O2, Py, rt, for 22′, 59%yield in two steps; iv) NaN₃, CAN, CH₃CN, −20° C.; v) PhSH, EtN(i-Pr)₂;Ccl₃CN, K₂CO₃; for 23′, 17% yield of 2:7,α/β in three steps; for 24′ 30%yield of 3;1, α/β in three steps; vi) LiBr, CH₃CN, for 25′, 46% yield, αonly; vii) Ac₂O, Py; Na-Hg, Na₂HPO₄, 94% yield in two steps, NaN₃, CAN,26% yield, PhSH, EtN(i-Pr)₂; K₂CO₃, Ccl₃CN, 53% yield in two steps(27′); viii) LiSph, THF, 60% yield, β only (26′).

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides novel α-O-linked glycoconjugates, usefulin the prevention and treatment of cancer.

The present invention provides a glycoconjugate having the structure:

A—B_(m)—C_(n)—D_(p)—E_(q)—F

wherein m, n, p and q are 0, 1, 2 or 3 such that m+n+p+q≦6; wherein A,B, C, D, E and F are independently amino acyl or hydroxy acyl residueswherein A is N- or O-terminal and is either a free amine or ammoniumform when A is amino acyl or a free hydroxy when A is hydroxy acyl, or Ais alkylated, arylated or acylated; wherein F is either a freecarboxylic acid, primary carboxamide, mono- or dialkyl carboxamide,mono- or diarylcarboxamide, linear or branched chain (carboxy)alkylcarboxamide, linear or branched chain (alkoxycarbonyl)alkyl-carboxamide,linear or branched chain (carboxy)arylalkylcarboxamide, linear orbranched chain (alkoxycarbonyl)alkylcarboxamide, an oligoester fragmentcomprising from 2 to about 20 hydroxy acyl residues, a peptidic fragmentcomprising from 2 to about 20 amino acyl residues, or a linear orbranched chain alkyl or aryl carboxylic ester; wherein from one to aboutfive of said amino acyl or hydroxy acyl residues are substituted by acarbohydrate domain having the structure:

wherein a, b, c, d, e, f, g, h, i, x, y and z are independently 0, 1, 2or 3; wherein the carbohydrate domain is linked to the respective aminoacyl or hydroxy acyl residue by substitution of a side group substituentselected from the group consisting of OH, COOH and NH₂; wherein R_(D) ishydrogen, a linear or branched chain alkyl, acyl, arylalkyl or arylgroup; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R⁹ are eachindependently hydrogen, OH, OR_(i), NH₂, NHCOR^(i), F, CH₂OH, CH₂OR^(i),a substituted or unsubstituted linear or branched chain alkyl, (mono,di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl oraryl group; wherein R^(i) is hydrogen, CHO, COOR^(ii), or a substitutedor unsubstituted linear or branched chain alkyl, arylalkyl or aryl groupor a saccharide moiety having the structure:

wherein Y and Z are independently NH or O; wherein k, l, r, s, t, u, vand w are each independently 0, 1 or 2, wherein R′₀ is hydrogen, alinear or branched chain alkyl, acyl, arylalkyl or aryl group; whereinR₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ are each independently hydrogen, OH,OR^(iii), NH₂, NHCOR^(iii), F, CH₂OH, CH₂OR^(iii), or a substituted orunsubstituted linear or branched chain alkyl, (mono-, di- ortri)hydroxyalkyl, (mono- di- or tri-)acyloxyalkyl, arylalkyl or arylgroup; wherein R₁₆ is hydrogen, COOH, COOR^(ii), CONHR^(ii), asubstituted or unsubstitued linear or branched chain alkyl or arylgroup; wherein R^(iii) is hydrogen, CHO, COOR^(iv), or a substituted orunsubstituted linear or branched chain alkyl, arylalkyl or aryl group;and wherein R^(ii) and R^(iv) are each independently H, or a substitutedor unsubstituted linear or branched chain alkyl, arylalkyl or arylgroup.

In a certain embodiment, the present invention provides theglycoconjugate as shown above wherein at least one carbohydrate domainhas the oligosaccharide structure of a cell surface epitope. In aparticular embodiment, the present invention provides the glycoconjugatewherein the epitope is Le^(a), Le^(b), Le^(x), or Le^(y). In anotherparticular embodiment, the present invention provides the glycoconjugatewherein the epitope is MBr1, a truncated MBr1 pentasaccharide or atruncated MBr1 tetrasaccharide.

In another embodiment, the present invention provides a glycoconjugatewherein the amino acyl residue is derived from a natural amino acid. Inanother embodiment, the invention provides the glycoconjugate wherein atleast one amino acyl residue has the formula: —NH—Ar—CO—. In a specificembodiment, the Ar moiety is P-phenylene.

In another embodiment, the present invention provides the glycoconjugatewherein at least one amino acyl or hydroxy acyl residue has thestructure:

wherein M, N and P are independently 0, 1 or 2; X is NH or O; Y is OH,NH or COOH; and wherein R′ and R″ are independently hydrogen, linear orbranched chain alkyl or aryl. In a specific embodiment, the amino acylresidue attached to the carbohydrate domain is Ser or Thr.

In another embodiment, the present invention provides the glycoconjugatewherein one or more of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁,R₁₂, R₁₃, R₁₄ and R₁₅ is 1RS,2RS,3-trihydroxy-propyl.

The present invention also provides a pharmaceutical composition fortreating cancer comprising the above-shown glycoconjugate and apharmaceutically suitable carrier.

The present invention further provides a method of treating cancer in asubject suffering therefrom comprising administering to the subject atherapeutically effective amount of the above-shown glycoconjugate and apharmaceutically suitable carrier. The method of treatment is effectivewhen the cancer is a solid tumor or an epithelial cancer.

The present invention also provides a trisaccharide having thestructure:

wherein R₁, R₃, R₄, R₅, R₆ and R₇ are each independently hydrogen, OH,OR^(i), NH₂, NHCOR^(i), F, N₃, CH₂OH, CH₂OR^(i), a substituted orunsubstituted linear or branched chain alkyl, (mono-, di- ortri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or arylgroup; wherein R^(i) is H, CHO, COOR^(ii), or a substituted orunsubstituted linear or branched chain alkyl, arylalkyl or aryl group;wherein R₂ is hydrogen, a linear or branched chain alkyl, acyl,arylalkyl or aryl group; wherein R₈ is hydrogen, COOH, COOR^(ii), CONHR^(ii), a substituted or unsubstituted linear or branched chain alkyl oraryl group; wherein R^(ii) is a substituted or unsubstituted linear orbranched chain alkyl, arylalkyl or aryl group; and wherein X is ahalide, a trihaloacetamidate, an alkyl or aryl sulfide or adialkylphosphite. In a preferred embodiment, the invention provides theabove-shown trisaccharide wherein X is a triethylphosphite. Theinvention further provides the trisaccharide wherein R₇ is1RS,2RS,3-trihydroxypropyl or 1RS,2RS,3-triacetoxypropyl. In addition,the invention provides the trisaccharide wherein R₈ is COOH.

The present invention also provides a trisaccharide amino acid havingthe structure:

wherein R₁, R₃, R₄, R₅, R₆ and R₇ are each independently hydrogen, OH,OR^(i), NH₂, NHCOR^(i), F, N_(3,) CH₂OH, CH₂OR^(i), a substituted orunsubstituted linear or branched chain alkyl, (mono-, di- ortri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or arylgroup; wherein R^(i) is H, CHO, COOR^(ii), or a substituted orunsubstituted linear or branched chain alkyl, arylalkyl or aryl group;wherein R₂ is hydrogen, a linear or branched chain alkyl, acyl,arylalkyl or aryl group; wherein R₈ is hydrogen, COOH, COOR^(ii),CONHR^(ii), a substituted or unsubstituted linear or branched chainalkyl or aryl group; wherein R^(ii) is a substituted or unsubstitutedlinear or branched chain alkyl, arylalkyl or aryl group; wherein R₀ is abase-labile N-protecting group; and wherein R′ is hydrogen or a loweralkyl group. A variety of N-protecting groups would be acceptable in thepreparation of the above-shown trisaccharide amino acid. R₀ maypreferably be one of several base-sensitive protecting groups, but morepreferably fluorenylmethyloxycarbonyl (FMOC).

The present invention provides a method of inducing antibodies in ahuman subject, wherein the antibodies are capable of specificallybinding with human tumor cells, which comprises administering to thesubject an amount of the glycoconjugate disclosed herein effective toinduce the antibodies. In a certain embodiment, the present inventionprovides a method of inducing antibodies wherein the glycoconjugate isbound to a suitable carrier protein. In particular, preferred examplesof the carrier protein include bovine serum albumin, polylysine or KLH.

In another embodiment, the present invention contemplates a method ofinducing antibodies which further comprises co-administering animmunological adjuvant. In a certain embodiment, the adjuvant isbacteria or liposomes. Specifically, favored adjuvants includeSalmonella minnesota cells, bacille Calmette-Guerin or QS21. Theantibodies induced are typically selected from the group consisting of(2,6)-sialyl T antigen, Le^(a), Le^(b), Le^(x), Le^(y), GM1, SSEA-3 andMBrI antibodies. The method of inducing antibodies is useful in caseswherein the subject is in clinical remission or, where the subject hasbeen treated by surgery, has limited unresected disease.

The present invention also provides a method of preventing recurrence ofepithelial cancer in a subject which comprises vaccinating the subjectwith the glycoconjugate shown above which amount is effective to induceantibodies. In practicing this method, the glycoconjugate may be usedalone or be bound to a suitable carrier protein. Specific examples ofcarrier protein used in the method include bovine serum albumin,polylysine or KLH. In a certain embodiment, the present method ofpreventing recurrence of epithelial cancer includes the additional stepof co-administering an immunological adjuvant. In particular, theadjuvant is bacteria or liposomes. Favored adjuvants include Salmonellaminnesota cells, bacille Calmette-Guerin or QS21. The antibodies inducedby the method are selected from the group consisting of (2,6)-sialyl Tantigen, Le^(a), Le^(b), Le^(x), Le^(y), GM1, SSEA-3 and MBrlantibodies.

The present invention further provides a glycoconjugate having thestructure:

wherein X is O or NR; wherein R is H, linear or branched chain alkyl oracyl; wherein A, B and C independently linear or branched chain alkyl oracyl, —CO—(CH₂)_(p)—OH or aryl, or have the structure:

wherein Y is O or NR; wherein D and E have the structure: —(CH₂)_(p)OHor —CO(CH₂)_(p)OH; wherein n and p are independently an integer between0 and 12; wherein D and E and, A, B, and C when they are —CO(CH₂)_(p)OH,are independently substituted by a carbohydrate domain having thestructure:

wherein a, b, c, d, e, f, g, h, i, x, y and z are independently 0, 1, 2or 3; wherein the carbohydrate domain is linked to the respectivehydroxy acyl residue by substitution of a terminal OH substituent;wherein R₀ is hydrogen, a linear or branched chain alkyl, acyl,arylalkyl or aryl group; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are each independently hydrogen, OH, OR^(i), NH₂, NHCOR^(i), F, CH₂OH,CH₂OR^(i), a substituted or unsubstituted linear or branched chainalkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl,arylalkyl or aryl group; wherein R^(i) is hydrogen, CHO, COOR^(ii), or asubstituted or unsubstituted linear or branched chain alkyl, arylalkylor aryl group or a saccharide moiety having the structure:

wherein Y and Z are independently NH or O; wherein k, l, r, s, t, u, vand w are each independently 0, 1 or 2, wherein R′₀ is hydrogen, alinear or branched chain alkyl, acyl, arylalkyl or aryl group; whereinR₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ are each independently hydrogen, OH,OR^(iii), NH₂, NHCOR^(iii), F, CH₂OH, CH₂OR^(iii), or a substituted orunsubstituted linear or branched chain alkyl, (mono-, di- ortri)hydroxyalkyl, (mono- di- or tri-)acyloxyalkyl, arylalkyl or arylgroup; wherein R₁₆ is hydrogen, COOH, COOR^(ii), CONHR^(ii), asubstituted or unsubstitued linear or branched chain alkyl or arylgroup; wherein R^(iii) is hydrogen, CHO, COOR^(iv), or a substituted orunsubstituted linear or branched chain alkyl, arylalkyl or aryl group;and wherein R^(ii) and R^(iv) are each independently H, or a substitutedor unsubstituted linear or branched chain alkyl, arylalkyl or arylgroup. In a certain embodiment, the present invention provides theabove-shown glycoconjugate wherein at least one carbohydrate domain hasthe oligosaccharide structure of a cell surface epitope. In oneembodiment, the epitope is Le^(a), Le^(b), Le^(x), or Le^(y). In anotherembodiment, the epitope is MBr1, a truncated MBr1 pentasaccharide or atruncated MBr1 tetrasaccharide. in a particular embodiment, theinvention provides the glycoconjugate shown above wherein one or more ofR₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ is1RS,2RS,3-trihydroxy-propyl.

The invention also provides a pharmaceutical composition for treatingcancer comprising the glycoconjugate shown above and a pharmaceuticallysuitable carrier.

The invention further provides a method of treating cancer in a subjectsuffering therefrom comprising administering to the subject atherapeutically effective amount of the glycoconjugate shown above and apharmaceutically suitable carrier. The method is useful in cases wherethe cancer is a solid tumor or an epithelial cancer.

The present invention also provides a glycoconjugate comprising a corestructure and a carbohydrate domain wherein the core structure is:

wherein M is an integer from about 2 to about 5,000; wherein N is 1, 2,3 or 4; wherein A and B are suitable polymer termination groups,including linear or branch chain alkyl or aryl groups; wherein the corestructure is substituted by the carbohydrate domain having thestructure:

wherein a, b, c, d, e, f, g, h, i, x, y and z are independently 0, 1, 2or 3; wherein the carbohydrate domain is linked to the core structure bysubstitution of the OH substituents; wherein R₀ is hydrogen, a linear orbranched chain alkyl, acyl, arylalkyl or aryl group; wherein R₁, R₂, R₃,R₄, R₅, R₆, R₇, R₈ and R₉ are each independently hydrogen, OH, OR_(i),NH₂, NHCOR^(i), F, CH₂OH, CH₂OR^(i), a substituted or unsubstitutedlinear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-,di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein R^(i) ishydrogen, CHO, COOR^(ii), or a substituted or unsubstituted linear orbranched chain alkyl, arylalkyl or aryl group or a saccharide moietyhaving the structure:

wherein Y and Z are independently NH or O; wherein k, l, r, s, t, u, vand w are each independently 0, 1 or 2, wherein R′₀ is hydrogen, alinear or branched chain alkyl, acyl, arylalkyl or aryl group; whereinR₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ are each independently hydrogen, OH,OR^(iii), NH₂, NHCOR^(iii), F, CH₂OH, CH₂OR^(iii), or a substituted orunsubstituted linear or branched chain alkyl, (mono-, di- ortri)hydroxyalkyl, (mono- di- or tri-)acyloxyalkyl, arylalkyl or arylgroup; wherein R₁₆ is hydrogen, COOH, COOR^(ii), CONHR^(ii), asubstituted or unsubstitued linear or branched chain alkyl or arylgroup; wherein R^(iii) is hydrogen, CHO, COOR^(iv), or a substituted orunsubstituted linear or branched chain alkyl, arylalkyl or aryl group;and wherein R^(ii) and R^(iv) are each independently H, or a substitutedor unsubstituted linear or branched chain alkyl, arylalkyl or arylgroup.

In a specific embodiment, the present invention provides a method ofpreparing glycopeptides related to the mucin family of cell surfaceglycoproteins. Mucins are characterized by aberrant α-O-glycosidationpatterns with clustered arrangements of carbohydrates α-O-linked toserine and threonine residues. FIG. 1. Mucins are common markers ofepithelial tumors (e.g., prostate and breast carcinomas) and certainblood cell tumors. Finn, O. J., et al., Immunol. Rev. 1995, 145, 61.

The (2,6)-Sialyl T antigen (ST antigen) is an example of the“glycophorin family” of α-O-linked glycopeptides (FIG. 2). It isselectively expressed on myelogenous leukemia cells. Fukuda, M., et al.,J. Biol. Chem. 1986, 261, 12796. Saitoh, O., et al., Cancer Res. 1991,51, 2854. Thus, in a specific embodiment, the present invention providesa synthetic route to pentapeptide 1, which is derived from theN-terminus of CD43 (Leukosialin) glycoprotein. Pallant, A., et al.,Proc. Natl. Acad. Sci. USA 1989, 86, 1328.

In particular, the invention provides a stereoselective preparation ofα-O-linked (2,6)-ST glycosyl serine and threonine via a block approach.In addition, the present invention provides an O-linked glycopeptideincorporating such glycosyl units with clustered ST epitopes (1,20).

A broad range of carbohydrate domains are contemplated by the presentinvention. Special mention is made of the carbohydrate domains derivedfrom the following cell surface epitopes and antigens:

MBr1 Epitope: Fucα1→2Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glu→0cer

Truncated MBr1 Epitope Pentasaccharide:

Fucα1→2Galβ1→3GalNAcβ1→3Galα1→4Galβ1

Truncated MBr1 Epitope Tetrasaccharide: Fucα1-2Galβ1→3GalNAcβ1→3Galα1

SSEA-3 Antigen: 2Galβ1→3GalNAcβ1→3Galα1→4Galβ1

Le^(y) Epitope: Fucα1→2Galβ1→4(Fucα1→3)GalNAcβ1

GM1 Epitope: Galβ1→3GalNAcβ1→4Galβ1→4(NeuAcα2→3)Glu→0cer

Methods for preparing carbohydrate domains based on a solid-phasemethodology have been disclosed in U.S. Pat. Nos. 5,543,505 and5,708,163 and in PCT International Application No. PCT/US96/10229, thecontents of which are incorporated by reference.

The present invention also provides a glycoconjugate having thestructure:

wherein m, n and p are integers between about 8 and about 20; wherein qis an integer between about 1 and about 8; wherein R_(V), R_(W), R_(X)and R_(Y) are independently hydrogen, optionally substituted linear orbranched chain lower alkyl or optionally substituted phenyl; whereinR_(A), R_(B) and R_(C) are independently a carbohydrate domain havingthe structure:

wherein a, b, c, d, e, f, g, h, i, x, y and z are independently 0, 1, 2or 3; wherein R₀ is hydrogen, linear or branched chain lower alkyl,acyl, arylalkyl or aryl group; wherein R₁,R₂, R₃, R₄, R₅, R₆, R₇, R₈ andR₉ are each independently hydrogen, OH, OR^(i), NH₂, NHCOR^(i), F,CH₂OH, CH₂OR^(i), an optionally substituted linear or branched chainlower alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- ortri)acyloxyalkyl, arylalkyl or aryl group; wherein R^(i) is hydrogen,CHO, COOR^(ii), or an optionally substituted linear or branched chainlower alkyl, arylalkyl or aryl group or a saccharide moiety having thestructure:

wherein Y and Z are independently NH or O; wherein k, l, r, s, t, u, vand w are each independently 0, 1 or 2, wherein R′₀ is hydrogen, alinear or branched chain alkyl, acyl, arylalkyl or aryl group; whereinR₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ are each independently hydrogen, OH,OR^(iii), NH₂, NHCOR^(iii), F, CH₂OH, CH₂OR^(iii), or a substituted orunsubstituted linear or branched chain alkyl, (mono-, di- ortri)hydroxyalkyl, (mono- di- or tri-)acyloxyalkyl, arylalkyl or arylgroup; wherein R₁₆ is hydrogen, COOH, COOR^(ii), CONHR^(ii), asubstituted or unsubstitued linear or branched chain alkyl or arylgroup; wherein R^(iii) is hydrogen, CHO, COOR^(iv), or a substituted orunsubstituted linear or branched chain alkyl, arylalkyl or aryl group;and wherein R^(ii) and R^(iv) are each independently H, or a substitutedor unsubstituted linear or branched chain alkyl, arylalkyl or arylgroup. In a certain embodiment, the invention provides a glycoconjugatewherein R^(v), R_(w), R_(x) and R_(y) are methyl.

In a certain other embodiment, the carbohydrate domains may beindependently monosaccharides or disaccharides. In one embodiment, theinvention provides a glycoconjugate wherein y and z are 0; wherein x is1; and wherein R₃ is NHAc. In another embodiment, the invention providesa glycoconjugate wherein h is 0; wherein g and i are 1; wherein R₇ isOH; wherein R₀ is hydrogen; and wherein R₈ is hydroxymethyl. In yetanother embodiment, m, n and p are 14; and wherein q is 3. In apreferred embodiment, each amino acyl residue of the glycoconjugatetherein has an L-configuration.

In a specific example, the carbohydrate domains of the glycoconjugateare independently:

In another example, the carbohydrate domains are independently

Additionally, the carbohydrate domains are independently:

The carbohydrate domains are also independently:

The carbohydrate domains also are independently

Also, the carbohydrate domains may be independently:

The carbohydrate domains are also independently:

The present invention provides a glycoconjugate having the structure:

wherein the carrier is a protein; wherein the cross linker is a moietyderived from a cross linking reagent capable of conjugating a surfaceamine of the carrier and a thiol; wherein m, n and p are integersbetween about 8 and about 20; wherein j and q are independently integersbetween about 1 and about 8; wherein R_(V), R_(W), R_(X) and R_(Y) areindependently hydrogen, optionally substituted linear or branched chainlower alkyl or optionally substituted phenyl; wherein R_(A), R_(B) andR_(C) are independently a carbohydrate domain having the structure:

wherein a, b, c, d, e, f, g, h, i, x, y and z are independently 0, 1, 2or 3; wherein R, is hydrogen, linear or branched chain lower alkyl,acyl, arylalkyl or aryl group; wherein R₁,R₂, R₃, R₄, R₅, R₆, R₇, R₈ andR₉ are each independently hydrogen, OH, OR^(i), NH₂, NHCOR^(i),F, CH₂OH,CH₂OR^(i),an optionally substituted linear or branched chain loweralkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl,arylalkyl or aryl group; wherein R^(i) is hydrogen, CHO, COOR^(ii),or anoptionally substituted linear or branched chain lower alkyl, arylalkylor aryl group or a saccharide moiety having the structure:

wherein Y and Z are independently NH or O; wherein k, l, r, s, t, u, vand w are each independently 0, 1 or 2, wherein R′₀ is hydrogen, alinear or branched chain alkyl, acyl, arylalkyl or aryl group; whereinR₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ are each independently hydrogen, OH,OR^(iii), NH₂, NHCOR^(iii), F, CH₂OH, CH₂OR^(iii), or a substituted orunsubstituted linear or branched chain alkyl, (mono-, di- ortri)hydroxyalkyl, (mono- di- or tri-)acyloxyalkyl, arylalkyl or arylgroup; wherein R₁₆ is hydrogen, COOH, COOR^(ii), CONHR^(ii), asubstituted or unsubstitued linear or branched chain alkyl or arylgroup; wherein R^(iii) is hydrogen, CHO, COOR^(iv), or a substituted orunsubstituted linear or branched chain alkyl, arylalkyl or aryl group;and wherein R^(ii) and R^(iv) are each independently H, or a substitutedor unsubstituted linear or branched chain alkyl, arylalkyl or arylgroup.

In one embodiment, the invention provides the glycoconjugate whereinR_(V), R_(W), R_(X) and R_(Y) are methyl. In another embodiment, theinvention provides the glycoconjugate wherein the carbohydrate domainsare monosaccharides or disaccharides. In another embodiment, theinvention provides the glycoconjugate wherein y and z are 0; wherein xis 1; and wherein R₃ is NHAc. In a further embodiment, the inventionprovides the glycoconjugate wherein h is 0; wherein g and i are 1;wherein R₇ is OH; wherein R₀ is hydrogen; wherein m, n and p are 14; andwherein q is 3; and wherein R₈ is hydroxymethyl.

In a certain embodiment, the invention provides the glycoconjugate asdisclosed wherein the protein is BSA or KLH. In a preferred embodiment,each amino acyl residue of the glycoconjugate has an L-configuration.

Specific examples of the glycoconjugate contain any of the followingcarbohydrate domains, which may be either the same or different in anyembodiment.

The present invention further provides a pharmaceutical composition fortreating cancer comprising a glycoconjugate as above disclosed and apharmaceutically suitable carrier.

The invention also provides a method of treating cancer in a subjectsuffering therefrom comprising administering to the subject atherapeutically effective amount of a glycoconjugate disclosed above anda pharmaceutically suitable carrier. In a certain embodiment, theinvention provides the method wherein the cancer is a solid tumor.Specifically, the method is applicable wherein the cancer is anepithelial cancer. Particularly effective is the application to treatprostate cancer.

The invention also provides a method of inducing antibodies in a humansubject, wherein the antibodies are capable of specifically binding withhuman tumor cells, which comprises administering to the subject anamount of the glycoconjugate disclosed above effective to induce theantibodies. In a certain embodiment, the invention provides the methodwherein the carrier protein is bovine serum albumin, polylysine or KLH.

In addition, the invention provides the related method of inducingantibodies which further comprises co-administering an immunologicaladjuvant. The adjuvant is preferably bacteria or liposomes. Inparticular, the adjuvant is Salmonella minnesota cells, bacilleCalmette-Guerin or QS21. The antibodies induced are favorably selectedfrom the group consisting of Tn, ST_(N), (2,3)ST, glycophorine,3-Le^(y), 6-Le^(y), T(TF) and T antibodies.

The invention further provides the method of inducing antibodies whereinthe subject is in clinical remission or, where the subject has beentreated by surgery, has limited unresected disease.

The invention also provides a method of preventing recurrence ofepithelial cancer in a subject which comprises vaccinating the subjectwith the glycoconjugate disclosed above which amount is effective toinduce antibodies. The method may be practiced wherein the carrierprotein is bovine serum albumin, polylysine or KLH. In addition, theinvention provides the related method of preventing recurrence ofepithelial cancer which further comprises co-administering animmunological adjuvant. Preferably, the adjuvant is bacteria orliposomes. Specifically, the preferred adjuvant is Salmonella minnesotacells, bacille Calmette-Guerin or QS21. The antibodies induced in thepractice of the methods are selected from the group consisting of Tn,ST_(N), (2,3)STN, glycophorine, 3-Le^(y), 6-Le^(y), T(TF) and Tantibodies.

The present invention also provides a method of preparing a protectedO-linked Le^(y) glycoconjugate having the structure:

wherein R is hydrogen, linear or branched chain lower alkyl oroptionally substituted aryl; R₁ is t-butyloxycarbonyl,fluorenylmethyleneoxycarbonyl, linear or branched chain lower alkyl oracyl, optionally substituted benzyl or aryl; R₂ is a linear or branchedchain lower alkyl, or optionally substituted benzyl or aryl; whichcomprises coupling a tetrasaccharide sulfide having the structure:

wherein R₃ is linear or branched chain lower alkyl or aryl; and R₄ ishydrogen, linear or branched chain lower alkyl or acyl, optionallysubstituted aryl or benzyl; or optionally substituted aryl sulfonyl;with an O-linked glycosyl amino acyl component having the structure:

under suitable conditions to form the protected O-linked Le^(y)glycoconjugate.

In one embodiment of the invention, the tetrasaccharide sulfide shownabove may be prepared by (a) halosulfonamidating a tetrasaccharideglycal having the structure:

under suitable conditions to form a tetrasaccharide halosulfonamidate;and (b) treating the halosulfonamidate with a mercaptan and a suitablebase to form the tetrasaccharide sulfide. In particular, the method maybe practiced wherein the mercaptan is a linear or branched chain loweralkyl or an aryl; and the base is sodium hydride, lithium hydride,potassium hydride, lithium diethylamide, lithium diisopropylamide,sodium amide, or lithium hexamethyldisilazide.

The invention also provides an O-linked glycoconjugate prepared by themethod disclosed.

In particular, the invention provides an O-linked glycopeptide havingthe structure:

wherein R₄ is a linear or branched chain lower acyl; and wherein R ishydrogen or a linear or branched chain lower alkyl or aryl. Variationsin the peptidic. portion of the glycopeptide are within the scope of theinvention. In a specific embodiment, the invention provides the O-linkedglycopeptide wherein R₄ is acetyl.

The present invention provides a method of preparing a protectedO-linked Le^(y) glycoconjugate having the structure:

wherein R is hydrogen, linear or branched chain lower alkyl oroptionally substituted aryl; R₁ is t-butyloxycarbonyl,fluorenylmethyleneoxycarbonyl, linear or branched chain lower alkyl oracyl, optionally substituted benzyl or aryl; and R₂ is a linear orbranched chain lower alkyl, or opionally subsituted benzyl or aryl;which comprises coupling a tetrasaccharide azidoimidate having thestructure:

with O-linked glycosyl amino acyl component having the structure:

under suitable conditions to form the protected O-linked Le^(y)glycoconjugate. The tetrasaccharide azidoimidate is favorably preparedby (a) treating a tetrasaccharide having the structure:

under suitable conditions to form an azido alcohol; and (b) reacting theazido alcohol with an imidoacylating reagent under suitable conditionsto form the azidoimidate. The tetrasaccharide azido nitrate may beprepared by (a) converting a tetrasaccharide glycal having thestructure:

under suitable conditions to form a peracylated tetrasaccharide glycalhaving the structure:

and (b) azidonitrating the glycal formed in step (a) under suitableconditions to form the tetrasaccharide azido nitrate. Step (b) isfavorably effected using cerium ammonium nitrate in the presence of anazide salt selected from the group consisting of sodium azide, lithiumazide, potassium azide, tetramethylammonium azide and tetraethylammoniumazide.

In addition, the invention provides an O-linked glycoconjugate preparedas shown above.

Once the carbohydrate domains covalently linked to O-bearing aminoacylside chains are prepared, the glycoconjugates of the subject inventionmay be prepared using either solution-phase or solid-phase synthesisprotocols, both of which are well-known in the art for synthesizingsimple peptides. Among other methods, a widely used solution phasepeptide synthesis method useful in the present invention uses FMOC (or arelated carbamate) as the protecting group for the α-amino functionalgroup; ammonia, a primary or secondary amine (such as morpholine) toremove the FMOC protecting group and a substituted carbodiimide (such asN,N′-dicyclohexyl- or -diisopropylcarbodiimide) as the coupling agentfor the C to N synthesis of peptides or peptide derivatives in a properorganic solvent. Solution-phase and solid phase synthesis of O-linkedglycoconjugates in the N to C direction is also within the scope of thesubject invention.

For solid-phase synthesis, several different resin supports have beenadopted as standards in the field. Besides the original chloromethylatedpolystyrene of Merrifield, other types of resin have been widely used toprepare peptide amides and acids, including benzhydrylamine andhydroxymethyl resins (Stewart, Solid Phase Peptide Synthesis,PierceChemical Co., 1984, Rockford, Ill.; Pietta, et al., J Chem. Soc. D.,1970, 650-651; Orlowski, et al, J. Org. Chem., 1976, 50, 3701-5;Matsueda et al, Peptides, 1981, 2, 45-50; and Tam, J. Org. Chem., 1985,50, 5291-8) and a resin consisting of a functionalizedpolystyrene-grafted polymer substrate (U.S. Pat. No. 5,258,454). Thesesolid phases are acid labile (Albericio, et al., Int. J. PeptideResearch. 1987, 30, 206-216). Another acid labile resin readilyapplicable in practicing the present invention uses atrialkoxydi-phenylmethylester moiety in conjunction with FMOC-protectedamino acids (Rink, Tetrahedron Letters, 1987, 28, 3787-90; U.S. Pat. No.4,859,736; and U.S. Pat. No. 5,004,781). The peptide is eventuallyreleased by cleavage with trifluoroacetic acid. Adaptation of themethods of the invention for a particular resin protocol, whether basedon acid-labile or base-sensitive N-protecting groups, includes theselection of compatible protecting groups, and is within the skill ofthe ordinary worker in the chemical arts.

The glycoconjugates prepared as disclosed herein are useful in thetreatment and prevention of various forms of cancer. Thus, the inventionprovides a method of treating cancer in a subject suffering therefromcomprising administering to the subject a therapeutically effectiveamount of any of the α-O-linked glycoconjugates disclosed herein,optionally in combination with a pharmaceutically suitable carrier. Themethod may be applied where the cancer is a solid tumor or an epithelialtumor, or leukemia. In particular, the method is applicable where thecancer is breast cancer, where the relevant epitope may be MBr1.

The subject invention also provides a pharmaceutical composition fortreating cancer comprising any of the α-O-linked glycoconjugatesdisclosed hereinabove, as an active ingredient, optionally thoughtypically in combination with a pharmaceutically suitable carrier. Thepharmaceutical compositions of the present invention may furthercomprise other therapeutically active ingredients.

The subject invention further provides a method of treating cancer in asubject suffering therefrom comprising administering to the subject atherapeutically effective amount of any of the α-O-linkedglycoconjugates disclosed hereinabove and a pharmaceutically suitablecarrier.

The compounds taught above which are related to α-O-linkedglycoconjugates are useful in the treatment of cancer, both in vivo andin vitro. The ability of these compounds to inhibit cancer cellpropagation and reduce tumor size in tissue culture will show that thecompounds are useful to treat, prevent or ameliorate cancer in subjectssuffering therefrom.

In addition, the glycoconjugates prepared by processes disclosed hereinare antigens useful in adjuvant therapies as vaccines capable ofinducing antibodies immunoreactive with various epithelial tumor andleukemia cells. Such adjuvant therapies may reduce the rate ofrecurrence of epithelial cancers and leukemia, and increase survivalrates after surgery. Clinical trials on patients surgically treated forcancer who are then treated with vaccines prepared from a cell surfacedifferentiation antigen found in patients lacking the antibody prior toimmunization, a highly significant increase in disease-free interval maybe observed. Cf. P. O. Livingston, et al., J. Clin. Oncol., 1994, 12,1036.

The magnitude of the therapeutic dose of the compounds of the inventionwill vary with the nature and severity of the condition to be treatedand with the particular compound and its route of administration. Ingeneral, the daily dose range for anticancer activity lies in the rangeof 0.001 to 25 mg/kg of body weight in a mammal, preferably 0.001 to 10mg/kg, and most preferably 0.001 to 1.0 mg/kg, in single or multipledoses. In unusual cases, it may be necessary to administer doses above25 mg/kg.

Any suitable route of administration may be employed for providing amammal, especially a human, with an effective dosage of a compounddisclosed herein. For example, oral, rectal, topical, parenteral,ocular, pulmonary, nasal, etc., routes may be employed. Dosage formsinclude tablets, troches, dispersions, suspensions, solutions, capsules,creams, ointments, aerosols, etc.

The compositions include compositions suitable for oral, rectal, topical(including transdermal devices, aerosols, creams, ointments, lotions anddusting powders), parenteral (including subcutaneous, intramuscular andintravenous), ocular (ophthalmic), pulmonary (nasal or buccalinhalation) or nasal administration. Although the most suitable route inany given case will depend largely on the nature and severity of thecondition being treated and on the nature of the active ingredient. Theymay be conveniently presented in unit dosage form and prepared by any ofthe methods well known in the art of pharmacy.

In preparing oral dosage forms, any of the unusual pharmaceutical mediamay be used, such as water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, and the like in the case of oral liquidpreparations (e.g., suspensions, elixers and solutions); or carrierssuch as starches, sugars, microcrystalline cellulose, diluents,granulating agents, lubricants, binders, disintegrating agents, etc., inthe case of oral solid preparations are preferred over liquid oralpreparations such as powders, capsules and tablets. If desired, capsulesmay be coated by standard aqueous or non-aqueous techniques. In additionto the dosage forms described above, the compounds of the invention maybe administered by controlled release means and devices.

Pharmaceutical compositions of the present invention suitable for oraladministration may be prepared as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient in powder or granular form or as a solution or suspension inan aqueous or nonaqueous liquid or in an oil-in-water or water-in-oilemulsion. Such compositions may be prepared by any of the methods knownin the art of pharmacy. In general compositions are prepared byuniformly and intimately admixing the active ingredient with liquidcarriers, finely divided solid carriers, or both and then, if necessary,shaping the product into the desired form. For example, a tablet may beprepared by compression or molding, optionally with one or moreaccessory ingredients. Compressed tablets may be prepared by compressingin a suitable machine the active ingredient in a free-flowing form suchas powder or granule optionally mixed with a binder, lubricant, inertdiluent or surface active or dispersing agent. Molded tablets may bemade by molding in a suitable machine, a mixture of the powderedcompound moistened with an inert liquid diluent.

The present invention will be better understood from the ExperimentalDetails which follow. However, one skilled in the art will readilyappreciate that the specific methods and results discussed are merelyillustrative of the invention as described in the claims which followthereafter. It will be understood that the processes of the presentinvention for preparing α-O-linked glycoconjugates encompass the use ofvarious alternate protecting groups known in the art. Those protectinggroups used in the disclosure including the Examples below are merelyillustrative.

Experimental Details General Procedures

All air- and moisture-sensitive reactions were performed in aflame-dried apparatus under an argon atmosphere unless otherwise noted.Air-sensitive liquids and solutions were transferred via syringe orcanula. Wherever possible, reactions were monitored by thin-layerchromatography (TLC). Gross solvent removal was performed in vacuumunder aspirator vacuum on a Buchi rotary evaporator, and trace solventwas removed on a high vacuum pump at 0.1-0.5 mmHg.

Melting points (mp) were uncorrected and performed in soft glasscapillary tubes using an Electrothermal series IA9100 digital meltingpoint apparatus. Infrared spectra (IR) were recorded using aPerkin-Elmer 1600 series Fourier-Transform instrument. Samples wereprepared as neat films on NaCl plates unless otherwise noted. Absorptionbands are reported in wavenumbers (cm⁻¹). Only relevant, assignablebands are reported.

Proton nuclear magnetic resonance (¹H NMR) spectra were determined usinga Bruker AMX-400 spectrometer at 400 MHz. Chemical shifts are reportedin parts per million (ppm) downfield from tetramethylsilane (TMS; δ=0ppm) using residual CHCl₃ as a lock reference (δ=7.25 ppm).Multiplicities are abbreviated in the usual fashion: s=singlet;d=doublet; t=triplet; q=quartet; m=multiplet; br=broad. Carbon nuclearmagnetic resonance (¹³C NMR) spectra were performed on a Bruker AMX-400spectrometer at 100 MHz with composite pulse decoupling. Samples wereprepared as with ¹H NMR spectra, and chemical shifts are reportedrelative to TMS (0 ppm) ; residual CHCl₃ was used as an internalreference (δ=77.0 ppm). All high resolution mass spectral (HRMS)analyses were determined by electron impact ionization (EI) on a JEOLJMS-DX 303HF mass spectrometer with perfluorokerosene (PFK) as aninternal standard. Low resolution mass spectra (MS) were deter-mined byeither electron impact ionization (El) or chemical ionization (Cl) usingthe indicated carrier gas (ammonia or methane) on a Delsi-Nermag R-10-10mass spectrometer. For gas chromatography/mass spectra (GCMS), a DB-5fused capillary column (30 m, 0.25 mm thickness) was used with helium asthe carrier gas. Typical conditions used a temperature program from60-250° C. at 40° C./min.

Thin layer chromatography (TLC) was performed using precoated glassplates (silica gel 60, 0.25 mm thickness). Visualization was done byillumination with a 254 nm UV lamp, or by immersion in anisaldehydestain (9.2 mL p-anisaldehyde in 3.5 mL acetic acid, 12.5 mL conc.sulfuric acid and 338 mL 95.% ethanol (EtOH)) and heating tocolorization. Flash silica gel chromatography was carried out accordingto the standard protocol.

Unless otherwise noted, all solvents and reagents were commercial gradeand were used as received, except as indicated hereinbelow, wheresolvents were distilled under argon using the drying methods listed inparentheses: CH₂Cl₂ (CaH₂); benzene (CaH₂); THF (Na/ketyl); Et,O(Na/ketyl); diisopropylamine (CaH₂).

Abbreviations TLC thin layer chromatography EtOAc ethyl acetate TIPStriisopropylsilyl PMB p-methoxybenzyl Bn benzyl Ac acetate hex hexaneTHF tetrahydrofuran coll collidine LiHMDS lithium hexamethyldisilazideDMF N,N-dimethylformamide DMAP 2-dimethylaminopyridine DDQ2,3-dichloro-5,6-dicyano-1,4-benzoquinone TBAF tetra-n-butylammoniumfluoride M.S. molecular sieves r.t. room temperature r.b. round bottomflask

EXAMPLE 12,6-Di-O-acetyl-3,40-carbonyl-β-D-galactopyranosyl-(1-3)-6-O-(triisopropylsilyl)-4-acetyl-galactal(3)

Galactal 2 (1.959 g, 9.89 mmol, 1.2 eq.) was dissolved in 100 mL ofanhydrous CH₂Cl₂ and cooled to 0° C. Solution of dimethyldioxirane (200mL of ca 0.06M solution in acetone) was added via cannula to thereaction flask. After 1 hr the starting material was consumed as judgedby TLC. Solvent was removed with a stream of N₂ and the crude epoxidewas dried in vacuo for 1 hr at room temperature. The crude residue(single spot by TLC) was taken up in 33 mL of THF and6-O-triisopropyl-galactal acceptor (2.50 g, 8.24 mmol) in 20 mL THF wasadded. The resulting mixture was cooled to −78° C. and ZnCl₂ (9.8 mL of1M solution in ether) was added dropwise. The reaction was slowly warmedup to rt and stirred overnight. The mixture was diluted with EtOAc andwashed with sat. sodium bicarbonate, then with brine and finally driedover MgSO₄. After evaporation of the solvent the crude material waspurified by flash chromatography (40-45-50-60% EtOAc/hexane) to yieldpure product which was immediately acetylated. 3.36 g was dissolved in50 mL of dry CH₂Cl₂, triethylamine (19.2 mL), cat amount of DMAP (ca 20mg) were added and the solution was cooled to 0° C. Acetic anhydride(9.9 mL) was added dropwise at 0° C. The reaction was stirred at rtovernight. The solvent was removed in vacuo and the crude material waschromatographed (50% EtOAc/hexane) to give glycal 3 (3.3 g, 75%): ¹H NMR(500 MHz, CDCl₃) δ6.42 (d, j=6.3 Hz, 1 H, H-1, glycal), 4.35 (½ AB, dd,j=6.8 Hz, 11.5 Hz, 1H, H-6′a), 4.28 (½AB, dd, j=6.1, 11.5 Hz, 1H,H-6′b).

EXAMPLE 22,6-Di-O-acetyl-3,4-O-carbonyl-β-D-galactopyranosyl-(1-3)-4-O-acetyl-galactal(4)

Compound 3 (1.5 g, 2.43 mmol) was dissolved in 24 mL of THF and cooledto 0° C. A mixture of TBAF (5.8 mL, 5.83 mmol, 2.4 eq.) and acetic acid(336 mL, 2.4 eq.) was added to the substrate at 0° C. The reaction wasstirred at 30° C. for 5 hrs. The reaction mixture was diluted with ethylacetate and quenched with sat sodium bicarbonate. Organic phase waswashed with sat sodium bicarbonate, brine and subsequently dried overmagnesium sulphate. The crude product was purified by chromatography(80-85-90% EtOAc/ hexane) to yield compound 4 (0.9 g, 80%): ¹H NMR (500MHz, CDCl₃) δ6.38 (dd, j=1.8, 6.3 Hz, 1 H, H-1, glycal), 5.39 (m, 1H,H-4), 2.22 (s, 3H, acetate), 2.16 (s, 3H, acetate), 2.13 (s, 3H,acetate).

EXAMPLE 3 [(Methyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-O-glycero-α-D-galacto-2-nonulopyranosylonate)-(2-6)]-(2,6-di-O-acetyl-3,4-O-carbonyl-β-D-galactopyranosyl)-(1-3)-4-O-acetyl-galactal.(6)

A flame dried flask was charged with sialyl phosphite donor 5 (69 mg,0.11 mmol, 1.3 eq.) and acceptor 4 (40 mg, 0.085 mmol) in the dry box(Argon atmosphere). The mixture was dissolved in 0.6 mL of dry THF. 0.6mL of dry toluene was added and the solution was slowly cooled to −60°C. to avoid precipitation. Trimethylsilyl triflate (2.4 μL, 0.11 eq.)was added and the mixture was stirred at −45° C. The reaction wasquenched at −45° C. after 2 hrs (completion judged by TLC) with 2 mL ofsat. sodium bicarbonate, warmed until water melted and the mixture waspoured into an excess of ethyl acetate. Organic layer was washed withsat. sodium bicarbonate and dried over anhydrous sodium sulphate. ¹H NMRof the crude material revealed a 4:1 ratio of α:β isomers (66.4 mg,84%). The mixture was separated by flash chromatography on silica gel(2-2.5-3-3.5-4% MeOH/CH₂Cl₂) to yield compound 6 (50 mg, 63% yield): ¹HNMR (500 MHz, CDCl₃) δ6.42 (d, j=6.2 Hz, 1H), 5.37 (m, 1H), 5.32-5.29(m, 4H), 5.26-5.24 (m, 1H), 5.12-5.10 (m, 2H), 4.98 (d, j=3.5 Hz, 1H),4.92-4.85 (m, 1H), 4.83-4.80 (m, 3H), 4.54 (m, 1H), 4.45 (dd, J=3.0,13.5 Hz, 1H), 4.33-4.20 (m, 3H), 4.22-4.02 (m, 7H), 3.96 (dd, j=7.6,10.9 Hz, 1H, H-2), 2.59 (dd, J=4.6, 12.9 Hz, 1H, H-2e NeuNAc), 2.30 (dd,J=12.9 Hz, 1H, H-2ax NeuNAc), 2.16, 2.14, 2.13, 2.12, 2.06, 2.03, 2.02(s, 7×3H, acetates), 1.88 (s, 3H, CH3CONH); FTIR (neat) 2959.2 (C—H),1816.5, 1745.0 (C═O), 1683.6, 1662.4 (glycal C═C), 1370.6, 1226.9,1038.7; HRMS (El) calc. for C39H51NO25K (M+K) 972.2386, found 972.2407.

EXAMPLE 4 α/β Mixture of azidonitrates 7

Compound 6 (370 mg, 0.396 mmol) was dissolved in 2.2 mL of dryacetonitrile and the solution was cooled to −20° C. Sodium azide (NaN₃,38.6 mg, 0.594, 1.5 eq.) and cerium ammonium nitrate (CAN, 651.3, 1.188mmol, 3eq.) were added and the mixture was vigorously stirred at −15° C.for 12 hrs. The heterogeneous mixture was diluted with ethyl acetate,washed twice with ice cold water and dried over sodium sulphate toprovide 400 mg of the crude product. Purification by flashchromatography provided mixture 7 (246 mg, 60% yield): ¹H NMR (400 MHz,CDCl₃) 6.35 (d, j=4.2 Hz, 1H, H-1, α-nitrate), 3.79 (s, 3H, methylester), 3.41 (dd, J=4.7, 11.0, 1H, H-2), 2.54 (dd, J=4.6, 12.8, H-2eqNeuNAc); FTIR (neat) 2117.4 (N3), 1733.9 (C═O); MS (El) calc. 1037.8,found 1038.4 (M+H).

EXAMPLE 5 α-Azidobromide 8

A solution of the compound 7 (150 mg, 0.145 mmol) in 0.6 mL of dryacetonitrile was mixed with lithium bromide (62.7 mg, 0.725 mmol, 5eq.)and stirred at rt for 3 hrs in the dark. The heterogeneous mixture wasdiluted with dichloromethane and the solution was washed twice withwater, dried over magnesium sulphate and the solvent was evaporatedwithout heating. After flash chromatography (5% MeOH, CH₂Cl₂) α-bromide8 (120 mg, 75% yield) was isolated and stored under an argon atmosphereat −80° C: ¹H NMR (500 MHz, CDCl₃) δ6.54 (d, J=3.7 Hz, 1H, H-1), 3.40(dd, J=4.5, 10.8 Hz, 1H, H-2), 2.57 (dd,J=4.5, 12.9, 1H, H-2eq NeuNAc),2.20, 2.15, 2.14, 2.12, 2.04, 2.02 (singlets, each 3H, acetates), 1.87(s, 3H, CH3CONH); MS (El) calc. for C39HS1N4BrO25 1055.7, found 1057.4(M+H).

EXAMPLE 6 Azido-trichloroacetamidate 9

Compound 7 (600 mg, 0.578 mmol) was dissolved in 3.6 mL of acetonitrileand the resulting solution was treated with thiophenol (180 μL) anddiisopropylethylamine (100 μL). After 10 minutes the solvent was removedwith a stream of nitrogen. The crude material was purified bychromatography (2-2.5-3-3.5% MeOH/CH₂Cl₂) to provide 472 mg (82%) ofintermediate hemiacetal. 60 mg (0.06 mmol) of this intermediate wastaken up in 200 mL of CH₂Cl₂ and treated with trichloroacetonitrile (60μL) and 60 mg potassium carbonate. After 6 hrs the mixture is dilutedwith CH₂C₂, solution is removed with a pipette and the excess K₂CO₃ waswashed three times with CH₂Cl₂. After evaporation of solvent the crudewas purified by flash chromatography (5%MeOH/CH₂Cl₂) to provide 9 (53.2mg, 64% yield for two steps, 1:1 mixture of α/β anomers). The anomerscan be separated by flash chromatography using a graded series ofsolvent systems (85-90-95-100% EtOAc/hexane).

EXAMPLE 7 Preparation of glycosyl-L-threonine 13 by AgClO₄-promotedglycosidation with glycosyl bromide 8

A flame dried flask is charged with silver perchlorate (27.3 mg, 2 eq),115 mg of 4 Å molecular sieves and N-FMOC-L-threonine benzyl ester (37.3mg, 0.086 mmol, 1.2 eq) in the dry box. 0.72 mL of CH₂Cl₂ was added tothe flask and the mixture was stirred at rt for 10 minutes. Donor 8 (76mg, 0.072 mmol) in 460 μL of CH₂Cl₂ was added slowly over 40 minutes.The reaction was stirred under argon atmosphere at rt for two hours. Themixture was then diluted with CH₂Cl₂ and filtered through celite. Theprecipitate was thoroughly washed with CH₂Cl₂, the filtrate wasevaporated and the crude material was purified on a silica gel column(1-1.5-2-2.5% MeOH/CH₂Cl₂) to provide 13 (74 mg, 74% yield). Theundesired δ-anomer was not detected by ¹H NMR and HPLC analysis of thecrude material. 13: ¹H NMR (500 MHz, CDCl₃) δ7.77 (d, J 7.5 Hz, 2H),7.63 (d, J 7.2 Hz, 2H), 7.40-7.25 (m, 8H), 5.72 (d, 9.2 Hz, 1H), 5.46(s, 1H), 5.33 (m, 1H), 5.29 (d, j=8.2 Hz, 1H), 5.23 (s, 2H), 5.11-5.04(m, 3H), 4.87-4.71 (m, 4H), 4.43-4.39 (m, 3H), 4.33-4.25 (m, 4H),4.09-3.97 (m, 6H), 3.79 (s, 3H, methyl ester), 3.66 (dd, j=3.7, 10.6 Hz,1H, H-3), 3.38 (dd, J=3.0, 10.7 Hz, 1H, H-2), 2.52 (dd, J=4.3, 12.7, 1H,H-2eq NeuNAc), 2.20, 2.13, 2.11, 2.10, 2.04, 2.03, 2.02 (singlets, 3H,acetates), 1.87 (s, 3H, CH3CONH), 1.35 (d, J=6.15 Hz, Thr-CH₃); FTIR(neat) 2110.3 (N3), 1748.7 (C═O), 1223.9, 1043.6; HRMS (El) calc. forC65H75N5O30K (M+K) 1444.4130, found 1444.4155.

EXAMPLE 8 Glycosyl-L-serine 12 BF₃.OEt₂ promoted glycosydation withtrichloroacetamidate 9

A flame dried flask is charged with donor 9 (50 mg, 0.044 mmol), 80 mgof 4 Å molecular sieves and N-FMOC-L-serine benzyl ester (27.5 mg, 0.066mmol) in the dry box. 0.6 mL of THF was added to the flask and themixture was cooled to −30° C. BF₃OEt₂ (2.8 mL, 0.022 mmol, 0.5 eq.) wasadded and the reaction was stirred under argon atmosphere. During threehours the mixture was warmed to −10° C. and then diluted with EtOAc andwashed with sat sodium bicarbonate while still cold. The crude materialwas purified on silica gel column (2-2.5-3% MeOH/CH₂Cl₂) to provide 12(40 mg, 66% yield) as a 4:1 mixture of α:β isomers. The pure α-anomerwas separated by flash chromatography (80-85-90-100% EtOAc/ hexane).

EXAMPLE 9 Glycosyl-L-threonine (15)

Compound 13 (47 mg, 33.42 μmol) was treated with thiolacetic acid (3 mL,distilled three times) for 27 hrs at rt. Thiolacetic acid was removedwith a stream of nitrogen, followed by toluene evaporation (four times).The crude product was purified by flash chromatography (1.5-2-2.5-3-3.5%MeOH/CH₂Cl₂) to yield 37 mg (78%) of an intermediated which wasimmediately dissolved in 7.6 mL of methanol and 0.5 mL of water. Afterpurging the system with argon 6.5 mg of palladium catalyst (100% Pd-C)was added and hydrogen balloon was attached. After 8 hrs hydrogen wasremoved by argon atmosphere, the catalyst was removed by filtrationthrough filter paper and the crude material was obtained upon removal ofsolvent. Flash Chromatography (10% MeOH/CH₂Cl₂) provided pure compound15 ( 36 mg, 78%): ¹H NMR (500 MHz, CDCl₃) mixture of rotamers,characteristic peaks δ3.80 (s, 3H, methyl ester), 3.41 (m, 1H, H-2),2.53 (m, 1H, H-2e NeuNAc)), 1.45 (d, J=5.1 Hz, Thr-CH₃), 1.35 (d, J=5.8Hz, Thr-CH3); FTIR (neat) 1818.2, 1747.2 (C═O), 1371.1, 1225.6, 1045.0;HRMS (El) calc. for C60H73N3O31K (M+K) 1370.3870, found 1370.3911.

EXAMPLE 10 Glycosyl-L-serine (14)

The compound 14 was prepared in 80% yield from 12 following the sameprocedure as for 15.

EXAMPLE 11 General Procedure for Peptide Coupling

Glycosyl amino acid 14 or 15 (1eq) and the peptide with a free aminogroup (1.2 eq) were dissolved in CH₂Cl₂ (22 mL/1 mmol). The solution wascooled to 0° C. and IIDQ (1.15-1.3 eq.) is added (1 mg in ca 20 mLCH₂Cl₂). The reaction was then stirred at rt for 8 hrs. The mixture wasdirectly added to the silica gel column.

EXAMPLE 12 General Procedure for FMOC Deprotection

A substrate (1 mmol in 36 mL DMF) was dissolved in anhydrous DMFfollowed by addition of KF (10 eq) and 18-crown-6 ether (catalyticamount). The mixture was then stirred for 48 hrs at rt. Evaporation ofDMF in vacuo was followed by flash chromatography on silica gel.

EXAMPLE 13 Glycopeptide 16

¹H NMR (500 MHz, CDCl₃) δ3.45-3.30 (m, 3×1 H, H-2), 3.74 (s, 3H, methylester), 2.58-2.49 (m, 3×1 H, H-2eq NeuNAc); FTIR (neat) 2961.7, 1819.2,1746.5, 1663.5, 1370.5, 1225.7, 1042.5; MS (El) calc. 3760, found 1903.8/ doubly charged=3806 (M+2Na).

EXAMPLE 14 Glycopeptide 1

¹HNMR (500 MHz, D₂O) d 4.73 (m, 2H, 2×H-1), 4.70 (d, 1 H, H-1), 4.64 (m,3H, 3×H-1′), 4.26-4.20 (m, 5H), 4.12-4.00 (m, 7H), 3.95-3.82 (7H),3.77-3.27 (m, 51H), 2.55-2.51 (m, 3H, 3×H-2eq NeuNAc), 1.84-1.82 (m,21H, CH3CONH), 1.52-1.45 (m, 3H, H-2ax NeuNAc), 1.20 (d, J=7.2 Hz, 3H),1.18 (d, J=6.6 Hz, 3H), 1.12 (d, J=6.2 Hz, 3H), 0.71 (d, j=6.6 Hz, 6H,val); 13C NMR (500 MHz, D2O) anomeric carbons: 105.06, 105.01, 100.60,100.57, 100.53, 100.11, 99.52, 98.70; MS (FAB) C96H157N11O64 2489 (M+H);MS(MALDI) 2497.

EXAMPLE 15

Glycopeptide 19

MS (El) calc. for C178H249N15O94Na2 4146 (M+2Na), found 4147, negativeionization mode confirmed the correct mass; MALDI (Matrix Assisted LaserDesorption Ionization) provided masses 4131, 4163.

EXAMPLE 16 Glycopeptide 20

MS(FAB) C119H193N15O70N 2975(M+Na)

EXAMPLE 17 Preparation of Azidonitrates 4′

To a solution of protected galactal 3′ (4.14 g, 12.1 mmol) in 60 ml ofanhydrous CH₃CN at −20 ° C. was added a mixture of NaN₃ (1.18 g, 18.1mmol) and CAN (19.8 g, 36.2 mmol). The reaction mixture was vigorouslystirred at −20° C. for overnight. Then the reaction mixture was dilutedwith diethyl ether, and washed with cold water and brine subsequently.Finally, the solution was dried over anhydrous Na₂SO₄. After evaporationof the solvent, the residue was separated by chromatography on silicagel. A mixture of α- and β-isomers (4′) (2.17 g, 40% yield) wasobtained. The ratio of α-isomer and β-isomer was almost 1:1 based on ¹HNMR. 4a′: [α]_(D) ²⁰ 94.5° (c 1.14, CHCl₃); FT-IR (film) 2940, 2862,2106, 1661, 1460, 1381, 1278 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ6.34 (d,J=3.9 Hz, 1H), 4.34(m, 2H), 4.21 (t, j=6.4 Hz, 1H), 3.95 (dd, J=9.6,7.2Hz, 1H), 3.85 (dd, J=9.6, 6.4 Hz, 1H), 3.78 (m, 1H), 1.52 (s, 3H),1.35 (s, 3H), 1.04 (m, 21H), ¹³C NMR (75 MHz, CDCl₃) δ110.29, 97.02,73.36, 71.89, 71.23, 61.95, 59.57, 28.18, 25.96, 17.86, 11.91; HRMS(FAB)calc. for C₁₈H₃₄N₄O₇SiK [M+K⁺] 485.1833, found 485.1821.

4b′: [α]_(D) ²⁰ 27.9° (c 1.28, CHCl₃); FT-IR (film) 2940, 2862, 2106,1666, 1459, 1376, 1283 cm⁻¹; ¹H NMR (300 MHz, CDC₁₃) δ5.50 (d, J=8.9 Hz,1H), 4.30 (dd, J=4.3, 1.5 Hz, 1H), 4.15 (dd, J=6.2, 4.3 Hz, 1H),3.89-4.03 (m, 3H), 3.56 (dd, J=8.9, 7.3Hz, 1H), 1.58 (s, 3H), 1.38 (s,3H), 1.08 (m, 21H); ¹³C NMR (75 MHz, CDCl₃) δ110.90, 98.09, 77.53,74.58, 71.99, 61.82, 61.68, 28.06, 25.97, 17.85, 11.89; HRMS (FAB) calc.for C₁₈H₃₄N₄O₇SiK [M+K⁺] 485.1833, found 485.1857.

EXAMPLE 18

Preparation of trichloroacetimidates 5a′ and 5b′

To a solution of a mixture of azidonitrates (4′) (1.36 g, 3.04 mmol) in10 ml of anhydrous CH₃CN at 0° C. were slowly added Et(i-Pr)₂N (0.53 ml,3.05 mmol) and PhSH (0.94 ml, 9.13 mmol) subsequently. The reactionmixture was stirred at 0° C. for 1 hour, then the solvent was evaporatedat room temperature in vacuo. The residue was separated bychromatography on silica gel to give the hemiacetal (1.22 g, 99.8%yield). To a solution of this hemiacetal (603 mg, 1.50 mmol) in 15 ml ofanhydrous CH₂Cl₂ at 0° C. were added K₂CO₃ (1.04 g, 7.50 mmol) andCCl₃CN (1.50 ml, 15.20 mmol). The reaction mixture was stirred from 0°C. to room temperature for 5 hours. The suspension was filtered througha pad of celite and washed with CH₂Cl₂. The filtrate was evaporated andthe residue was separated by chromatography on silica gel togive_α-trichloroacetimidate 5a′ (118 mg, 14% yield),β-trichloroacetimidate 5b′ (572 mg, 70% yield) and recovered hemiacetal(72 mg). 5a′: [α]_(D) ²⁰ 84.0° (c 1.02, CHCl₃); FT-IR (film) 2942, 2867,2111, 1675, 1461, 1381, 1244 cm⁻¹; ¹H NMR (300 MHz, CDC₁₃) δ8.69 (s,1H), 6.29 (d, J=3.3 Hz, 1H), 4.47 (dd, J=8.0, 5.3 Hz, 1H), 4.39 (dd,j=5.3, 2.4Hz, 1H), 4.25 (m, 1H), 3.97 (dd, J=9.5, 7.8 Hz, 1H), 3.87 (dd,j=9.5, 6.0 Hz, 1H), 3.67 (dd, j=8.0, 3.3 Hz, 1H), 1.53 (s, 3H), 1.36 (s,3H), 1.04 (m, 21 H); ¹³C NMR (75 MHz, CDCl₃) δ160.67, 109.98, 94.72,77.20, 73.35, 72.11, 70.83, 62.01, 60.80, 28.29, 26.09,17.88, 11.88;HRMS (FAB) calc. for C₂₀H₃₅N₄O₅SiKCl₃ [M+K⁺] 583.1080, found 583.1071.

5b′: [α]_(D) ²⁰ 30.6° (c 1.12, CHCl₃); FT-IR (film) 2941, 2110, 1677,1219 cm⁻¹; ¹H NMR (300 MHz, CDC₁₃)_(—)δ8.71 (s, 1H), 5.57 (d, J=9.0 Hz,1H), 4.27 (d, j=5.2Hz, 1H), 3.95-4.02 (m, 4H), 3.63 (t, j=9.0 Hz, 1H).1.57 (s, 3H), 1.34 (s, 3H), 1.04 (m, 21 H); ^(13C NMR ()75 MHz, CDCl₃)δ160.94, 110.55, 96.47, 77.20, 74.58, 72.21, 64.84, 61.89, 28.29, 26.07,17.87, 11.90; HRMS (FAB) calc. for C₂₀H₃₅N₄O₅SiKC₃ [M+K⁺] 583.1080,found 583.1073.

EXAMPLE 19 Preparation of Glycosyl Fluorides 6a′ and 6b′

To a solution of the hemiacetal prepared previously (68.0 mg, 0.169mmol) in 3 ml of anhydrous CH₂Cl₂ at 0° C. was added DAST (134 ml, 1.02mmol) slowly. The reaction mixture was stirred at 0° C. for 1 hour. Thenthe mixture was diluted with EtOAc, washed with sat. NaHCO₃ and brinesubsequently. Finally, the solution was dried over anhydrous Na₂SO₄.After evaporation of the solvent, the residue was separated bychromatography on silica gel to give α-fluoride 6a′ (30.2 mg, 44% yield)and β-fluoride 6b′ (33.7 mg, 49% yield). 6a′: [α]_(D) ²⁰ 689.5° (c 1.47,CHCl₃); FT-IR (film) 2944, 2867, 2115, 1462, 1381 cm⁻¹; ¹H NMR (300 MHz,CDC₁₃) δ5.59 (dd,J=53.0, 2.6 Hz, 1H), 4.34-4.40 (m, 2H), 4.26 (m, 1H),3.96 (t, j=9.3 Hz, 1H), 3.88 (dd, J=9.3, 6.0 Hz, 1H), 3.48 (ddd, J=25.5,7.0, 2.6 Hz, 1H), 1.50 (s, 3H), 1.34 (s, 3H), 1.05 (m, 21H); ¹³C NMR (75MHz, CDCl₃) δ110.03, 107.45, 104.46, 77.21, 76.38, 73.21, 71.79, 70.48,61.88, 61.23, 60.91, 28.17, 26.03, 17.09, 11.92; HRMS (FAB) calc. forC₁₈H₃₅N₃O₄SiF [M+H⁺] 404.2378, found 404.2369.

6b″: [α]_(D)20 153.8° (c 1.65, CHCl₃);.FT-IR (film) 2943, 2867, 2116,1456, 1382, 1246 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ5.05 (dd, J=52.6, 7.4Hz, 1H), 4.27 (dt, J=5.5, 2.0 Hz, 1H), 3.89-4.05 (m, 4H), 3.70 (dt,j=12.3, 5.1 Hz, 1H), 1.53 (s, 3H), 1.32 (s, 3H), 1.04 (m, 21H); ¹³C NMR(75 MHz, CDCl₃) δ110.64, 109.09, 106.24 ,76.27, 76.16, 73.42, 71.63,64.80, 64.52, 61.77, 27.80, 25.78, 17.03, 11.86; HRMS (FAB) calc. forC₁₈H₃₅N₃O₄SiF [M+H⁺] 404.2378, found 404.2373.

EXAMPLE 20 Coupling of β-trichloroacetimidate 5b′ with protected serinederivative 7′: Synthesis of 9a′ and 9b′

To a suspension of β-trichloroacetimidate 5b′ (52.3 mg, 0.096 mmol),serine derivative 7′ (44.0 mg, 0.105 mmol) and 200 mg 4 Å molecularsieve in a mixture of 2 ml of anhydrous CH₂Cl₂ and 2 ml of anhydroushexane at −78° C. was added a solution of TMSOTf (1.91 μl, 0.01 mmol) in36 μl of CH₂Cl₂. The reaction mixture was stirred at −78° C. for a halfhour, then warmed up to room temperature for 3 hours. The reaction wasquenched by Et₃N. The suspension was filtered through a pad of celiteand washed with EtOAc. The filtrate was washed with H₂O, brine and driedover anhydrous Na₂SO₄. After evaporation of the solvent, the residue wasseparated by chromatography on silica gel to give α-product 9a′ (55 mg,71% yield) and β-product 9b′ (22 mg, 29% yield). 9a′: [α]_(D) ²⁰ 70.5°(c 2.0, CHCl₃); FT-IR (film) 3433, 3348, 2943, 2867, 2109, 1730, 1504,1453, 1381, 1336 cm⁻¹; ¹H NMR (300 MHz, CDC₁₃) δ7.74 (d, J=7.5 Hz, 2H),7.57 (d, J=7.5 Hz, 2H), 7.25-7.40 (m, 9H), 5.73 (d, J=8.4 Hz, 1H), 5.24(d, J=12.1 Hz, 1H), 5.17 (d, J=12.1, 1H), 4.73 (d, J=3.2 Hz, 1H), 4.60(m, 1H), 4.41 (dd, J=10.2, 7.2 Hz, 1H), 4.20-4.31 (m, 4H), 3.82-3.98 (m,5H), 3.23 (dd, J=8.0, 3.2 Hz, 1H), 1.47 (s, 3H), 1.31 (s, 3H), 1.02 (m,21H); ¹³C NMR (75 MHz, CDCl₃) δ169.65, 155.88, 143.81, 143.73, 141.27,135.04, 128.63, 128.54, 127.71, 127.60, 125.18, 125.11, 109.67, 98.71,77.23, 72.88, 72.39, 68.95, 68.79, 67.73, 67.36, 62.28, 61.10, 54.39,47.08, 28.26, 26.10, 17.91, 11.90; HRMS (FAB) calc. for C₄₃H₅₆N₄O₉SiK[M+K⁺] 839.3453, found 839.3466, 839.3453;

9b′: [α]_(D) ²⁰20.60 (c 1.05, CHCl₃); FT-IR (film) 3433, 2943,2866,2114, 1729, 1515, 1453, 1382 cm⁻¹; ¹H NMR (300 MHz, CDC₁₃) δ7.78 (d,J=7.4 Hz, 2H), 7.63 (t, J=7.4 Hz, 2H), 7.30-7.44 (m, 9H), 5.91 (d,J=8.4Hz, 1H), 5.30 (d, J=12.4 Hz, 1H), 5.26 (d, J=12.4 Hz, 1H), 4.65 (m,1H), 4.48 (dd, J=10.0, 2.6 Hz, 1H), 4.39 (t, J=7.4 Hz, 2H), 4.23-4.28(m, 3H), 3.89-4.04 (m, 3H), 3.85 (dd, J=10.0, 3.1 Hz, 1H), 3.78 (m, 1H),3.41 (t, J=8.2 Hz, 1H), 1.58 (s, 3H), 1.36 (s, 3H), 1.08 (m, 21H); ¹³CNMR (75 MHz, CDCl₃) δ169.37, 155.92, 143.90, 143.69, 141.25, 135.27,128.55, 128.27, 127.94, 127.68, 127.07, 125.27, 125.21, 119.94, 110.37,102.30, 76.87, 73.78, 72.19, 69.68, 67.40, 67.33, 65.44, 61.99, 54.20,47.06, 28.32, 26.10, 17.89, 11.88; HRMS (FAB) calc. for C₄₃H₅₆N₄O₉SiK[M+K³⁰ ] 839.3453, found 839.3466.

EXAMPLE 21 Coupling of β-trichloroacetimidate 5b′ with protected serinederivative 7′ in THF Promoted by TMSOTf (0.5 eq.)

To a suspension of trichloroacetimidate 5b′ (14.4 mg, 0.027 mmol),serine derivative 7′ (16.7 mg, 0.040 mmol) and 50 mg 4 Å molecular sievein 0.2 ml of anhydrous THF at −78° C. was added a solution of TMSOTf(2.7 μl, 0.013 mmol) in 50 μl of THF. The reaction was stirred at −78°C. for 2 hours and neutralized with Et₃N. The reaction mixture wasfiltered through a pad of celite and washed with EtOAc. The filtrate waswashed with H₂O, brine and dried over anhydrous Na₂SO₄. Afterevaporation of the solvent, the residue was separated by chromatographyon silica gel to give the a-product 9a′ (18.5 mg, 86% yield).

EXAMPLE 22 Coupling of α-trichloroacetimidate 5a with protected serinederivative 7′ in THF Promoted by TMSOTf (0.5 eq.):

To a suspension of trichloroacetimidate 5a′ (12.3 mg, 0.023 mmol),serine derivative 7′ (14.1 mg, 0.034 mmol) and 50 mg 4 Å molecular sievein 0.2 ml of anhydrous THF at −78° C. was added a solution of TMSOTf(2.2 μl, 0.011 mmol) in 45 μl of THF. The reaction was stirred at −78°C. for 4 hours and neutralized with Et₃N. The reaction mixture wasfiltered through a pad of celite and washed with EtOAc. The filtrate waswashed with H₂O, brine and dried over anhydrous Na₂SO₄. Afterevaporation of the solvent, the residue was separated by chromatographyon silica gel to give the a-product 9a′ (11.8 mg, 66% yield).

EXAMPLE 23 Coupling of β-trichloroacetimidate 5b′ with protectedthreonine derivative 8: Synthesis of 10a′ and 10b′

To a suspension of β-trichloroacetimidate 5b′ (50.6 mg, 0.093 mmol),threonine derivative 8′ (44.0 mg, 0.102 mmol) and 200 mg 4Å molecularsieve in a mixture of 2 ml of anhydrous CH₂Cl₂ and 2 ml of anhydroushexane at −78° C. was added a solution of TMSOTf (1.85 μl, 0.009 mmol)in 35 μl of CH₂Cl₂. The reaction mixture was stirred at −78° C. for ahalf hour, then warmed up to room temperature for 4 hours. The reactionwas quenched by Et₃N. The suspension was filtered through a pad ofcelite and washed with EtOAc. The filtrate was washed with H₂O, brineand dried over anhydrous Na₂SO₄. After evaporation of the solvent, theresidue was separated by chromatography on silica gel to give recoveredthreonine derivative 7′ (28.0 mg), the α-product 10a′ (22.0 mg, 29%yield) and the β-product 10b′ (3.0 mg, 4% yield). 10a′: [α]_(D) ²⁰ 55.2°(c 0.88, CHCl₃); FT-IR (film) 3430, 2941, 2866, 2109, 1730, 1510, 1452,1380 cm⁻¹; ¹H NMR (300 MHz, CDC₁₃) δ7.75 (d, J=7.5 Hz, 2H), 7.59 (d,J=7.5 Hz, 2H), 7.26-7.41 (m, 9H), 5.62 (d, J=9.4 Hz, 1 H), 5.22 (d,J=12.3 Hz, 1 H), 5.18 (d, J=1 2.3 Hz, 1H), 4.73 (d, J=3.6 Hz, 1H),4.36-4.47 (m, 3H), 4.19-4.32 (m, 4H), 4.09 (m, 1H), 3.91 (dd, J=9.8, 6.6Hz, 1H), 3.83 (dd, J=9.8, 5.5 Hz, 1H), 3.24 (dd, J=8.1, 3.6 Hz, 1H),1.49 (s, 3H), 1.33 (s, 3H), 1.32 (d, J=6.0 Hz, 3H), 1.05 (m, 21H); ¹³CNMR (75 MHz, CDCl₃) 6170.12, 156.74, 143.94, 143.69, 141.29, 135.00,128.65, 128.59, 127.70, 127.10, 125.19, 119.96, 109.78, 99.09, 77.22,73.16, 72.53, 69.03, 67.71, 67.40, 62.54, 61.61, 58.84, 47.15, 28.32,26.17, 18.76, 17.94, 11.92; HRMS (FAB) calc. for C₄₄H₅₈N₄O₉SiK [M+K⁺]853.3608, found 853.3588;

10b′: [α]_(D) ²⁰ 92.4° (c 0.47, CH₂Cl₂); FT-IR (film) 3434, 3351, 2940,2865, 2111, 1728, 1515, 1455 cm⁻¹; ¹H NMR (300 MHz, CDC₁₃) δ7.74 (d,J=7.5 Hz, 2H), 7.59 (t, J=7.5 Hz, 2H). 7.25-7.40 (m, 9H), 5.68 (d, J=9.3Hz, 1H), 5.20 (d, J=12.4 Hz, 1H), 5.17 (d, J=12.4 Hz, 1H), 4.58 (m, 1H),4.47 (dd, J=9.3, 3.4 Hz, 1H), 4.34 (d, J=7.8 Hz, 2H), 4.18-4.29 (m, 3H),3.96 (t, J=8.9 Hz, 1H), 3.84 (dd,J=10.0, 5.2 Hz, 1H), 3.81 (dd,J=8.2,5.2 Hz, 1H), 3.65 (m, 1H), 3.34 (t, J=8.1 Hz, 1H), 1.55 (s, 3H), 1.32(s, 3H), 1.30 (d, J=6.4 Hz, 3H), 1.02 (m, 21H); ¹³C NMR (75 MHz, CDCl₃)δ169.89, 156.73, 143.96, 143.73, 141.27, 135.38, 128.61, 128.27, 127.93,127.67, 127.08, 125.26, 119.93, 110.26, 99.32, 77.91, 77.82, 74.03,73.55, 72.01, 67.42, 67.25, 65.32, 61.66, 58.61, 47.12, 28.36,26.08,17.88, 16.52,11.87; HRMS(FAB) calc. for C₄₄H₅₈N₄O₉SiNa [M+Na⁺]837.3869, found 837.3887.

EXAMPLE 24 Coupling of α-glycosyl fluoride 6a′ with protected threoninederivative 8′ in CH₂Cl₂ promoted by (Cp)₂ZrCl₂—AgClO₄

To a suspension of AgClO₄ (25.1 mg, 0.121 mmol), (Cp)₂ZrCl₂ (17.8 mg,0.06 mmol) and 150 mg 4 Å molecular sieve in 1 ml of anhydrous CH₂Cl₂ at−30° C. was added a solution of β-glycosyl fluoride 6a′ (16.3 mg, 0.04mmol) and threonine derivative 8′ (19.2 mg, 0.045 mmol) in 4.0 ml ofanhydrous CH₂Cl₂ slowly. The reaction was stirred at −30° C. for 6 hoursand quenched with sat. NaHCO₃. The solution was filtered through a padof celite and washed with EtOAc. The filtrate was washed with sat.NaHCO₃, brine and dried over anhydrous Na₂SO₄. After evaporation of thesolvent, the residue was separated by chromatography on silica gel togive the α-product 10a′ (24.8 mg, 75% yield) and the β-product 10b′ (3.9mg, 12% yield).

EXAMPLE 25 Coupling of β-glycosyl fluoride 6b′ with protected threoninederivative 8′ in CH₂Cl₂ promoted by (Cp)₂ZrCl₂-AgClO₄

To a suspension of AgClO₄ (24.4 mg, 0.118 mmol), (Cp)₂ZrCl₂ (17.2 mg,0.059 mmol) and 200 mg 4 Å molecular sieve in 1 ml of anhydrous CH₂Cl₂at −30° C. was added a solution of β-glycosyl fluoride 6b′ (1 5.8 mg,0.03918 mmol) and threonine derivative 8′ (20.3 mg, 0.04702 mmol) in 4.0ml of anhydrous CH₂Cl₂ slowly. The reaction was stirred at −30° C. for10 hours and quenched with sat. NaHCO₃. The solution was filteredthrough a pad of celite and washed with EtOAc. The filtrate was washedwith sat. NaHCO₃, brine and dried over anhydrous Na₂SO₄. Afterevaporation of the solvent, the residue was separated by chromatographyon silica gel to give the α-product 10a′ (22.3 mg, 70% yield) and theβ-product 10b′ (3.9 mg, 12% yield).

EXAMPLE 26 Deprotection of the silyl group of 9a′

To a solution of the α-product 9a′ (15.0 mg, 0.01873 mmol) in 2 ml ofTHF at 0° C. were added HOAc (56 μl, 0.978 mmol) and 1M TBAF (240 μl.0.240 mmol). The reaction was run at 0° C. for 1 hour, and then warmedup to room temperature for 3 days. The mixture was diluted with EtOAc,washed with H₂O, brine, and finally dried over anhydrous Na₂SO₄. Afterevaporation of the solvent, the residue was separated by chromatographyon silica gel to give desired product 11′ (12.4 mg, 100%). 11′:

[α]_(D) ²⁰ 78.3° (c 0.67, CH₂Cl₂); FT-IR (film) 3432, 3349, 2987, 2938,2109, 1729, 1517, 1452, 1382 cm⁻¹; ¹H NMR (300 MHz, CDC₁₃) δ7.75 (d,J=7.5 Hz, 2H), 7.59 (d, J=7.5 Hz, 2H), 7.27-7.41 (m, 9H), 6.01 (d, J=9.2Hz, 1H), 5.21 (d, J=12.4 Hz, 1H), 5.18 (d, J=12.4 Hz, 1H), 4.74 (d,J=3.3 Hz, 1H), 4.58 (m, 1H), 4.41 (d, J=7.0 Hz, 2H), 4.14-4.23 (m, 3H),4.02 (dd, J=5.4, 2.4 Hz, 1H), 3.91-3.97 (m, 2H), 3.68-3.85 (m, 2H), 3.27(dd, J=8.2, 3.3 Hz, 1H), 1.48 (s, 3H), 1.33 (s, 3H); ¹³C NMR (75 MHz,CDCl₃) δ169.71, 155.85, 143.78, 143.71, 141.32, 135.03, 128.59, 127.72,127.08, 125.08, 119.99, 110.20, 99.12, 77.20, 73.35, 73.11, 70.22,68.54, 67.76, 67.04, 62.48, 60.73, 54.66, 47.12, 28.10, 26.14; HRMS(FAB) calc. for C₃₄H₃₇N₄O₉ [M+H⁺] 645.2560, found 645.2549.

EXAMPLE 27 Deprotection of the silyl group of 10a′

To a solution of the α-product 10a′ (16.0 mg, 0.02 mmol) in 3 ml of THFat 0° C. were added HOAc (67 μl, 1.18 mmol) and 1M TBAF (300 μl, 0.3000mmol). The reaction was run at 0° C. for 1 hour, and then warmed up toroom temperature for 3 days. The mixture was diluted with EtOAc, washedwith H₂O, brine, and finally dried over anhydrous Na₂SO₄. Afterevaporation of the solvent, the residue was separated by chromatographyon silica gel to give desired product 12′ (12.1 mg, 94%). 12′

[α]_(D) ²⁰ 731.8° (c 0.62, CH₂Cl₂); FT-IR (film) 3430, 2986, 2936, 2109,1728, 1515, 1451, 1382 cm⁻¹; ¹H NMR (300 MHz, CDC₁₃) δ7.75 (d, J=7.4 Hz,2H), 7.60 (d, J=7.4 Hz, 2H), 7.25-7.41 (m, 9H), 5.67 (d, J=9.0 Hz, 1H),5.21 (br.s, 2H), 4.82 (d, J=3.2 Hz, 1H), 4.40-4.52 (m, 3H), 4.33-4.38(m, 2H), 4.19-4.29 (m, 2H), 4.09 (m, 1H), 3.75-3.92 (m, 2H), 3.30 (dd,J=8.0, 3.2 Hz, 1H), 2.04 (m, 1H), 1.50 (s, 3H), 1.35 (s, 3H), 1.30 (d,J=6.2 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ170.13, 156.69, 143.91, 143.69,141.30, 134.98, 128.61, 127.72, 127.10, 125.20, 119.97, 110.25, 98.39,76.26, 73.49, 68.35, 67.75, 67.36, 62.62, 61.31, 58.69, 47.16, 28.18,26.24, 18.54; HRMS (FAB) calc. for C₃₅H₃₉N₄O₉[M+H⁺] 659.2716, found659.2727.

EXAMPLE 28 Preparation of compound 14′

To a suspension of trichloroacetimidate 13′ (332.0 mg, 0.435 mmol), theacceptor 11′ (140.2 mg, 0.218 mmol) and 1.0 g 4 Å molecular sieve in 4ml of anhydrous CH₂Cl₂ at −30° C. was added a solution of BF₃.Et₂O (13.8μl, 0.109 mmol) in 120 μl of anhydrous CH₂Cl₂ slowly. The reactionmixture was stirred at −30° C. for overnight, then warmed up to roomtemperature for 3 hours. The reaction was quenched with Et₃N, filteredthrough a pad of celite and washed with EtOAc. The filtrate was washedwith H₂O, brine and dried over anhydrous Na₂SO₄. After evaporation ofthe solvent, the residue was separated by chromatography on silica gelto give crude recovered acceptor 11′ which was further converted tocompound 9a′ (87.0 mg, 0.109 mmol) and crude coupling product which wasfurther reduced to compound 14′ by pyridine and thiolacetic acid. Thecrude coupling product was dissolved in 1 ml of anhydrous pyridine and 1ml of thiolacetic acid at 0° C. The reaction mixture was stirred at roomtemperature for overnight. The solvent was evaporated in vacuo at roomtemperature and the residue was separated by chromatography on silicagel to give compound 14′ (99.6 mg, 72% yield based on 50% conversion ofacceptor 11′). 14′:

[α]_(D) ²⁰ 267.9° (c 4.0, CHCl₃); FT-IR (film) 3361, 3018, 1 751, 1672,1543, 1452, 1372 cm⁻¹; ¹H NMR (300 MHz, CDC₁₃) δ7.72 (d, J=7.5 Hz, 2H),7.58 (m, 2H), 7.26-7.38 (m, 9H), 6.26 (d, J=8.2 Hz, 1H), 5.83 (d, J=9.3Hz, 1H), 5.59 (d, J=9.2 Hz, 1H), 5.32 (d, J=2.7 Hz, 1H), 5.16 (s, 2H),5.02-5.11 (m, 2H), 4.94 (dd, J=10.4, 3.4 Hz, 1H), 4.59 (d, J=3.4 Hz,1H), 4.35-4.52 (m, 6H), 3.60-4.19 (m, 16H), 2.11 (s, 3H), 2.05 (s, 3H),2.02 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H), 1.93 (s, 3H), 1.91 (s, 3H),1.83 (s, 3H), 1.48(s, 3H), 1.24 (s, 3H); ¹³C NMR (75 MHz, CDCl₃)δ170.33, 170.23, 170.15, 170.07, 169.94, 169.85, 169.19, 155.92, 143.75,143.64, 141.22, 135.12, 128.62, 128.39, 127.67, 127.01, 124.99, 119.93,109.81, 101.12, 100.84, 98.14, 77.21, 75.49, 74.28, 72.61, 72.12, 70.74,69.10, 68.80, 67.61, 67.38, 67.28, 67.09, 66.64, 62.28. 60.77, 54.25,53.03, 50.09, 47.09, 27.76, 26.40, 23.18, 23.03, 20.71, 20.47, 20.36;HRMS(FAB) calc. for C₆₂H₇₅N₃O₂₆Na [M+Na⁺] 1300.4539, found 1300.4520 .

EXAMPLE 29 Preparation of Compound 15′

To a suspension of trichloroacetimidate 13′ (305.0 mg, 0.3996 mmol), theacceptor 12′ (131.6 mg, 0.1998 mmol) and 1.0 g 4 Å molecular sieve in 4ml of anhydrous CH₂Cl₂ at −30° C. was added a solution of BF₃.Et₂O (12.7μl, 0.10 mmol) in 115 μl of anhydrous CH₂Cl₂ slowly. The reactionmixture was stirred at −30° C. for overnight, then warmed up to roomtemperature for 3 hours. The reaction was quenched with Et₃N, filteredthrough a pad of celite and washed with EtOAc. The filtrate was washedwith H₂O, brine and dried over anhydrous Na₂SO₄. After evaporation ofthe solvent, the residue was separated by chromatography on silica gelto give crude recovered acceptor 12′ which was further converted tocompound 10a′ (85.0 mg, 0.104 mmol) and crude coupling product which wasfurther reduced to compound 15′ by pyridine and thiolacetic acid. Thecrude coupling product was dissolved in 1 ml of anhydrous pyridine and 1ml of thiolacetic acid at 0° C. The reaction mixture was stirred at roomtemperature for overnight. The solvent was evaporated in vacuo at roomtemperature and the residue was separated by chromatography on silicagel to give compound 15′ (71.1 mg, 58% yield based on 48% conversion ofacceptor 12′). 15′:

[α]_(D) ²⁰ 346.8° (c 0.53, CHCl₃); FT-IR (film) 3366, 2986, 1750, 1673,1541, 1452, 1372 cm⁻¹; ¹H NMR (300 MHz, CDC₁₃) δ7.73 (d, J=7.4 Hz, 1H),7.57 (d, J=7.4 Hz, 2H), 7.27-7.45 (m, 9H), 5.83 (d, J=9.4 Hz, 1H), 5.74(d, J=9.4 Hz, 1H), 5.61 (d, J=8.9 Hz, 1 H), 5.31 (d, J=3.0 Hz, 1H),4.91-5.16 (m, 5H), 4.62 (d, J=3.2 Hz, 1H), 4.32-4.46 (m, 6H), 3.95-4.22(m, 11H), 3.64-3.84 (m, 3H), 3.57 (m, 1H), 2.12 (s, 6H), 2.10 (s, 3H),2.06 (s, 3H), 2.01 (s, 6H), 1.93 (s, 3H), 1.86 (s, 3H), 1.51 (s, 3H),1.26 (s, 3H), 1.22 (d, J=5.5 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ170.70,170.38, 170.19, 169.94, 169.86, 169.74, 169.20, 156.34, 143.72, 143.59,141.26, 134.59, 128.74, 128.37, 127.71, 127.03, 124.92, 119.94, 109.76,101.48, 100.86, 99.48, 77.20, 76.23, 75.49, 74.41, 72.74, 72.43, 70.76,69.26, 69.13, 67.56, 67.45, 67.13, 66.65, 62.29, 60.78, 58.47, 52.83,50.35, 47.16, 27.86, 26.54, 23.22, 23.03, 20.72, 20.49,20.37, 18.20;HRMS (FAB) calc. for C₆₃H₇₈N₃O₂₆ [M+H⁺] 1292.4871, found 1292.4890.

EXAMPLE 30 Synthesis of Compound 1′

The trisaccharide 14′ (105.8 mg, 0.083 mmol) was dissolved in 5 ml of⁸⁰% aq. HOAc at room temperature. The reaction mixture was stirred atroom temperature for overnight, then at 40° C. for 3 hours. The solutionwas extracted with EtOAc, washed with sat. NaHCO₃, H₂O, brine, and driedover anhydrous Na₂SO₄. After evaporation of the solvent, the residue wasseparated by chromatography on silica gel to give diol (93.0 mg, 91%yield). To a solution of this diol (91.5 mg, 0.074 mmol) in 10 ml ofanhydrous CH₂Cl₂ at 0° C. were added catalytic DMAP (4.5 mg, 0.037mmol), Et₃N (103 μl, 0.74 mmol) and Ac₂O (28 μl, 0.30 mmol)subsequently. The reaction was run for overnight at room temperature.The reaction mixture was diluted with EtOAc, washed with H₂O, brine anddried over anhydrous Na₂SO₄. After evaporation of the solvent, theresidue was separated by chromatography on silica gel to giveperacetylated compound (88.8 mg, 91%yield). To a suspension of 10% Pd/C(5.0 mg) in a mixture of 1 ml of MeOH and 0.1 ml of H₂O was added asolution of the peracetylated compound (38.5 mg, 0.03 mmol) in 4.0 ml ofMeOH.

The reaction was stirred under H₂ atmosphere at room temperature for 4hours. The reaction mixture was passed through a short column of silicagel to remove the catalyst and washed with MeOH. After removal of thesolvent, the residue was dissolved in 1.5 ml of DMF and to this solutionwas added 0.5 ml of morpholine at 0° C. slowly. The reaction was stirredat room temperature for overnight. The solvent was evaporated in vacuoand the residue was separated by chromatography on silica gel to give29.0 mg material which was further deacetylated in basic condition. Thematerial got previously was dissolved in 50 ml of anhydrous THF and 5 mlof anhydrous MeOH. The solution was cooled to 0° C. and to this solutionwas added a solution of NaOMe (14.0 mg, 0.26 mmol) in 5 ml of anhydrousMeOH. The reaction was stirred at room temperature for overnight andquenched with 50% aq. HOAc. After evaporation of the solvent, theresidue was separated by chromatography on reverse-phase silica gel togive crude product, which was further purified by gel permeationfiltration on Sephadex LH-20 to give the final product 1′ (15.1 mg,77%yield). 1′: [α]_(D) ²⁰ 715.6° (c 0.1, H₂O); ¹H NMR (300MHz,CD₃OD—D₂O) δ4.85 (d, J=3.4 Hz, 1H), 4.55 (d, J=7.4 Hz, 1H), 4.46 (d,J=7.0 Hz, 1H), 4.26 (dd, J=10.9, 3.5 Hz, 1H), 3.34-4.09 (m, 20H), 2.07(s, 3H), 2.06 (s, 3H); ¹³C NMR (75 MHz, CD₃OD—D₂O) δ175.64, 175.36,104.61, 102.98, 99.57, 80.35, 76.94, 76.36, 74.32, 73.88, 72.57, 71.30,70.82, 70.16, 69.21, 62.50, 61.62, 56.64, 51.58, 51.22, 23.63, 23.40;HRMS(FAB) calc. for C₂₅H₄₄N₃O₁₈ [M+H⁺] 674.2620, found 674.2625.

EXAMPLE 31 Synthesis of Compound 2′

The trisaccharide 15′ (70.2 mg, 0.054 mmol) was dissolved in 5 ml of 80%aq. HOAc at room temperature. The reaction mixture was stirred at roomtemperature for overnight, then at 40° C. for 3 hours. The solution wasextracted with EtOAc, washed with sat. NaHCO₃, H₂O, brine, and driedover anhydrous Na₂SO₄. After evaporation of the solvent, the residue wasseparated by chromatography on silica gel to give diol (67.1 mg, 99%yield). To a solution of diol (65.1 mg, 0.052 mmol) in 8 ml of anhydrousCH₂Cl₂ at 0° C. were added catalytic DMAP (3.2 mg, 0.026 mmol), Et₃N (72μl, 0.52 mmol) and Ac₂O (20 μl, 0.21 mmol) subsequently. The reactionwas run for overnight at room temperature. The reaction mixture wasdiluted with EtOAc, washed with H₂O, brine and dried over anhydrousNa₂SO₄. After evaporation of the solvent, the residue was separated bychromatography on silica gel to give peracetylated compound (66.0 mg,95%yield). To a suspension of 10% Pd/C (5.0 mg) in a mixture of 1 ml ofMeOH and 0.1 ml of H₂O was added a solution of the peracetylatedcompound (22.1 mg, 0.017 mmol) in 4.0 ml of MeOH. The reaction wasstirred under H₂ atmosphere at room temperature for 4 hours. Thereaction mixture was passed through a short column of silica gel toremove the catalyst and washed with MeOH. After removal of the solvent,the residue was dissolved in 1.5 ml of DMF and to this solution wasadded 0.5 ml of morpholine at 0° C. slowly. The reaction was stirred atroom temperature for overnight. The solvent was evaporated in vacuo andthe residue was separated by chromatography on silica gel to give 29.0mg material which was further deacetylated in basic condition. Thematerial got previously was dissolved in 50 ml of anhydrous THF and 5 mlof anhydrous MeOH. The solution was cooled to 0° C. and to this solutionwas added a solution of NaOMe (14.9 mg, 0.276 mmol) in 5 ml of anhydrousMeOH. The reaction was stirred at room temperature for overnight andquenched with 50% aq. HOAc. After evaporation of the solvent, theresidue was separated by chromatography on reverse-phase silica gel togive crude product, which was further purified by gel permeationfiltration on Sephadex LH-20 to give the final product 2′ (8.4 mg,74%yield). 2′: [α]_(D) ²⁰ 418.4° (c 0.1, H₂O); ¹H NMR (300 MHz,CD₃O_(D)—D₂O) δ4.91 (d, J=3.3 Hz, 1H), 4.56 (d, J=8.2 Hz, 1H), 4.46 (d,J=7.4 Hz, 1H), 3.52-4.22 (m, 20H), 2.10 (s, 3H), 2.06 (s, 3H), 1.36 (d,J=6.5 Hz, 3H); ¹³C NMR (75 MHz, CD₃OD—D₂O) δ175.90, 175.48, 104.20,103.97, 102.47, 79.75, 78.71, 76.72, 76.56, 73.92, 73.76, 70.94, 70.52,70.10, 69.79, 68.98, 62.25, 61.28, 56.25, 51.20, 50.79, 23.51, 19.44;HRMS(FAB) calc. for C₂₆H₄₆N₃O₁₆ [M +H⁺] 688.2776, found 688.2774.

EXAMPLE 32 Preparation of Thioglycoside 17′

To a suspension of perbenzylated lactal 16′ (420 mg, 0.49 mmol) and 600mg of 4 Å molecular sieve in 5 ml of anhydrous CH₂Cl₂ was addedbenzenesulfonamide (116 mg, 0.74 mmol) at room temperature. After 10minutes, the suspension was cooled to 0° C. and l(sym-collidine)₂CIO₄was added in one portion. Fifteen minutes later, the solution wasfiltered through a pad of celite and washed with EtOAc. The organicsolution was washed with Na₂S₂O₃, brine and dried over Na₂SO₄. Afterevaporation of the solvent, the residue was separated by chromatographyon silica gel to give 500 mg of iodosulfonamidate derivative (90%yield).To a solution of ethanethiol (150 μl, 1.98 mmol) in 4 ml of anhydrousDMF at −40° C. was added a solution of LiHMDS (0.88 ml, 0.88 mmol).After 15 minutes, a solution of iodosulfonamidate (450 mg, 0.397 mmol)in 6 ml of anhydrous DMF was added slowly at that temperature. Thereaction mixture was stirred at −40° C. for 4 hours, and quenched withH₂O. The aqueous solution was extracted by EtOAc three times and thecombined organic layer was washed with H₂O, brine and dried over Na₂SO₄.After evaporation of the solvent, the residue was separated bychromatography on silica gel to give the desired thioglycoside 17′ (350mg, 83%yield) and recover the iodosulfonamidate (60 mg). 17′: IR (film)3020, 3000, 2860, 1480, 1450 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ7.87 (d,J=7.7 Hz, 2H), 7.17-7.45 (m, 33H), 5.01 (d, J=8.9 Hz, 1H), 4.93 (d,J=11.4 Hz, 1H), 4.79 (s, 2H), 4.69 (m, 3H), 4.56 (d, J=11.3 Hz, 2H),4.30-4.50 (m, 6H), 3.95 (t, J=5.0 Hz, 1H), 3.90 (d, J=2.7 Hz, 1H), 3.75(m, 3H), 3.65 (m, 2H), 3.52 (m, 2H), 3.39-3.46 (m, 3H), 2.50 (q, J=7.4Hz, 2H), 1.12 (t, J=7.4 Hz, 3H); HRMS(FAB) calc. for C₆₂H₆₇O₁₁NS₂K[M+K⁺] 1104.3789, found 1104.3760.

EXAMPLE 33 Preparation of Trisaccharide 20′

In a round-bottom flask were placed thioglycoside 17′ (2.10 g, 1.97mmol), acceptor 18′ (964 mg, 2.95 mmol), di-t-butylpyridine (2.65 ml,11.81 mmol) and 7.0 g of 4 Å molecular sieve. The mixture was dissolvedin 10 ml of anhydrous CH₂Cl₂ and 20 ml of anhydrous Et₂O. This solutionwas cooled to 0° C. and then MeOTf (1.1 1 ml, 8.85 mmol) was added to itslowly. The reaction mixture was stirred at 0° C. for overnight. Afterfiltration through a pad of celite, the organic layer was submitted toaqueous work-up. The EtOAc extraction was dried over Na₂SO₄. Afterevaporation of the solvent, the residue was separated by chromatographyon silica gel to give 20α′ (206 mg, 8%) and 20β′ (2.26 g, 86%). 20β′: IR(film) 3020, 3000, 2860, 1480, 1450 cm⁻¹; ¹H NMR (300MHz, CDCl₃) δ7.82(d, J=7.7 Hz, 2H), 7.20-7.45 (m, 43H), 6.32 (d, J=6.2 Hz, 1H), 4.96 (d,J=9.2 Hz, 1H), 4.90 (d, J=6.2 Hz, 1H), 4.80 (m, 4H), 4.72 (s, 2H),4.54-4.68 (m, 6H), 4.28-4.48 (m, 6H), 4.07 (br.s, 1H), 4.00 (t, J=5.0Hz, 1H), 3.90 (s, 1H), 3.74 (m, 4H), 3.35-3.61 (m, 10H); HRMS(FAB) calc.for C₈₀H₈₃O₁₅NSK [M+K⁺] 1368.5123, found 1368.5160.

EXAMPLE 34 Preparation of Trisaccharide 21′

In a round-bottom flask were placed thioglycoside 17′ (966 mg, 0.906mmol), acceptor 19′ (219 mg, 1.18 mmol), di-t-butylpyridine (1.22 ml,5.44 mmol) and 2.5 g of 4 Å molecular sieve. The mixture was dissolvedin 5 ml of anhydrous CH₂Cl₂ and 10 ml of anhydrous Et₂O. This solutionwas cooled to 0° C. and then MeOTf (0.51 ml, 4.53 mmol) was added to itslowly. The reaction mixture was stirred at 0° C. for 5 hours. Afterfiltration through a pad of celite, the organic layer was submitted toaqueous work-up. The EtOAc extraction was dried over Na₂SO₄. Afterevaporation of the solvent, the residue was separated by chromatographyon silica gel to give 21α′ (59 mg, 6%) and 21β′ (910 mg, 84%).

21a′: IR (film) 3020, 3000, 2860, 1480, 1450 cm⁻¹; ¹H NMR (300 MHz,CDCl₃) δ(7.83 (d, J=7.5 Hz, 2H), 7.12-7.46 (m, 33H), 6.36 (d,J=6.2 Hz,1H), 5.11 (d,J=8.9 Hz, 1H), 4.98 (d, J=10.9 Hz, 1H), 4.93 (d, J=11.6,1H), 4.83 (d, J=8.1 Hz, 1H), 4.80 (d, J=11.6 Hz, 1H), 4.68-4.73 (m, 4H),4.50-4.58 (m, 3H), 4.27-4.32 (m, 4H), 4.27 (d, J=6.2 Hz, 1H), 4.05 (m,1H), 3.97 (m, 2H), 3.83 (m, 2H), 3.70 (m, 2H), 3.58 (m, 2H), 3.24-3.49(m, 4H), 1.52 (s, 3H), 1.41 (s, 3H); HRMS(FAB) calc. for C₆₉H₇₅O₁₅NSNa[M+Na⁺] 1212.4756, found 1212.4720. 21β′: IR (film) 3020, 3000, 2860,1480, 1450 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ_(7.87 (d, J=7.2 Hz, 2H),7.19-7.45 (m, 33H), 6.35 (d, J=6.2 Hz, 1H), 4.98 (d, J=8.9 Hz, 1H), 4.95(d, J=11.6 Hz, 1H), 4.78 (m, 4H), 4.67 (m, 3H), 4.56 (m, 2H), 4.50 (d,J=12.0 Hz, 1H), 4.43 (d, J=6.2 Hz, 1H), 4.27-4.39 (m, 4H), 4.04 (d,J=6.2 Hz, 1H), 3.97 (t, J=7.2 Hz, 1H), 3.90 (d, J=2.5 Hz, 1H), 3.73-3.82(m, 3H), 3.48-3.66 (m, 6H), 3.35-3.42 (m, 3H), 1.43 (s, 3H), 1.30 (s,3H); HRMS(FAB) calc. for C₆₉H₇₅O₁₅NSNa [M +Na+] 1212.4755, found1212.4780.

EXAMPLE 35 Preparation of trisaccharide 22′

In a flame-dried flask was condensed 30 ml of anhydrous NH₃ at −78° C.To this liquid NH₃ was added sodium metal (320 mg, 13.95 mmol) in oneportion. After 15 minutes, the dry ice-ethanol bath was removed and thedark blue solution was refluxed for 20 minutes. It was cooled down to−78° C. again and a solution of trisaccharide 20′ (619 mg, 0.47 mmol) in6 ml of anhydrous THF was added slowly. The reaction mixture wasrefluxed at −30° C. for half hour and quenched with 10 ml of MeOH. Afterevaporation of NH₃, the basic solution was neutralized by Dowex resin.The organic solution was filtered and evaporated to give crude productwhich was submitted to acetylation. The crude product was dissolved in3.0 ml of pyridine and 2.0 ml of Ac₂O in the presence of 10 mg of DMAPat 0° C. The reaction mixture was stirred from 0° C. to room temperaturefor overnight. After aqueous work-up, the organic layer was dried overNa₂SO₄. The solvent was evaporated and the residue was separated bychromatography on silica gel to give peracetylated trisaccharide 22′(233 mg, 59%). 22′: [α]_(D) ²⁰ −19.77° (c 1.04, CHCl₃); IR(film) 1740,1360 cm⁻¹, ¹H NMR (300 MHz, CDCl₃) δ6.46 (dd, J=6.2, 1.5 Hz, 1H), 5.64(d, J=9.1 Hz, 1H), 5.54 (d, J=2.0 Hz, 1H), 5.40 (d, J=4.5 Hz, 1H), 5.36(d, J=2.9 Hz, 1H), 5.12 (m, 2H), 4.98 (dd, J=10.4, 3.4 Hz, 1H), 4.70 (d,J=6.2 Hz, 1H), 4.58 (d, J=7.3 Hz, 1H), 4.50 (m, 2H), 4.26 (t, J=5.0 Hz,1H), 4.12 (m, 3H), 3.89 (m, 2H), 3.78 (m, 2H), 3.64 (m, 1H), 2.16 (s,3H), 2.13 (s, 3H), 2.12 (s, 3H), 2.09 (s, 3H), 2.07 (s, 3H), 2.06 (s,3H), 2.05 (s, 3H), 2.02 (s, 3H), 1.98 (s, 3H); ¹³C NMR (75 MHz, CDCl₃)δ170.29, 170.14, 169.24, 145.34, 128.20, 100.85, 100.72, 88.86, 75.58,74.26, 72.58, 72.06, 70.71, 70.61, 68.98, 66.77, 66.55, 64.19, 63.53,62.09, 60.70, 52.97, 23.05, 20.72, 20.56; HRMS(FAB) calc. forC₃₆H₄₉O₂₂NNa [M+Na⁺] 870.2645, found 870.2644.

EXAMPLE 36 Preparation of trisaccharide donor 23′

To a solution of trisaccharide glycal 20′ (460 mg, 0.346 mmol) in 3 mlof anhydrous CH₃CN at −25° C. were added NaN₃ (34 mg, 0.519 mmol) andCAN (569 mg, 1.4 mmol) subsequently. The mixture was stirred at −25° C.for 8 hours. After aqueous work-up, the organic layer was dried overNa₂SO₄. The solvent was evaporated and the residue was separated bychromatography on silica gel to give a mixture of azidonitratederivatives (134 mg, 27%). This azidonitrate mixture was hydrolyzed inthe reductive condition. The azidonitrates was dissolved in 2 ml ofanhydrous CH₃CN at room temperature. EtN(i-Pr)₂ (16 μl, 0.091 mmol) andPhSH (28 μl, 0.272 mmol) were added subsequently. After 15 minutes, thereaction was complete and the solvent was evaporated at roomtemperature. The hemiacetal derivative (103 mg, 74%) was obtained afterchromatography on silica gel. This hemiacetal (95 mg, 0.068 mmol) wasdissolved in 2 ml of anhydrous CH₂Cl₂. To this solution were added 1 mlof CCl₃CN and 0.5 g of K₂CO₃ at room temperature. The reaction was runfor overnight. After filtration through a pad of celite, the organicsolvent was evaporated and the residue was separated by chromatographyon silica gel to give 23α′ (18 mg, 17%) and 23β′ (70 mg, 67%). 23α′: ¹HNMR (300 MHz, CDCl₃) δ8.71 (s, 1H), 7.96 (d, J=8.2 Hz, 2H), 6.92-7.50(m, 33H), 6.56 (d, J=2.8 Hz, 1H), 5.02 (m, 3H), 4.92 (d, J=11.6 Hz, 2H),4.86 (d, J=11.6 Hz, 1H), 4.22-4.64 (m, 18H), 3.95-4.07 (m, 3H), 3.85 (m,2H), 3.72 (m, 2H), 3.63 (m, 1H), 3.35-3.56 (m, 4H), 3.34 (dd, J=10.3,2.8 Hz, 1H).

23β′: ¹H NMR (300 MHz, CDCl₃) δ8.40 (s, 1H), 8.10 (d, J=8.1 Hz, 2H),6.90-7.45 (m, 33H), 6.37 (d, J=9.4 Hz, 1H), 5.93 (d, J=8.2 Hz, 1H), 5.04(d, J=11.6 Hz, 2H), 4.98 (d, J=11.6 Hz, 1H), 4.90 (d, J=11.7 Hz, 1H),4.83 (d, J=11.7 Hz, 1H), 4.79 (d, J=11.6 Hz, 1H), 4.77 (d, J=11.6 Hz,1H), 4.72 (d, J=8.2 Hz, 1H), 4.40-4.63 (m, 8H), 4.19-4.38 (m, 5H),3.86-4.10 (m, 6H), 3.63 (m, 2H), 3.42-3.50 (m, 4H), 3.35 (m, 2H), 3.25(d, J=9.1 Hz, 1H).

EXAMPLE 37 Preparation of Trisaccharide Donor 24′

To a solution of trisaccharide glycal 21′ (225 mg, 0.264 mmol) in 2 mlof anhydrous CH₃CN at −15° C. were added NaN₃ (26 mg, 0.40 mmol) and CAN(436 mg, 0.794 mmol) subsequently. The mixture was stirred at −15° C.for overnight. After aqueous work-up, the organic layer was dried overNa₂SO₄. The solvent was evaporated and the residue was separated bychromatography on silica gel to give a mixture of azidonitratederivatives (130 mg, 51%). This azidonitrate mixture was hydrolyzed inthe reductive condition. The azidonitrates (125 mg, 0.129 mmol) wasdissolved in 5 ml of anhydrous CH₃CN at room temperature. EtN(i-Pr)₂ (25μl, 0.147 mmol) and PhSH (45 μl, 0.441 mmol) were added subsequently.After 15 minutes, the reaction was complete and the solvent wasevaporated at room temperature. The hemiacetal derivative (92 mg, 77%)was obtained after chromatography on silica gel. This hemiacetal (80 mg,0.087 mmol) was dissolved in 5 ml of anhydrous CH₂Cl₂. To this solutionwere added 0.9 ml of CCl₃CN and 0.12 g of K₂CO₃ at room temperature. Thereaction was run for overnight. After filtration through a pad ofcelite, the organic solvent was evaporated and the residue was separatedby chromatography on silica gel to give a mixture of α and β isomer of24′ (71 mg, 77%, α:β 3:1). 24′: ¹H NMR (300MHz, CDCl₃) δ9.55 (s, 1 H, NHof β isomer), 8.71 (s, 1 H, NH of α isomer), 6.54 (d, J=3.6 Hz, amomericH of α isomer)

EXAMPLE 38 Preparation of Trisaccharide Donor 25′

The azidonitrate derivatives (100 mg, 0.103 mmol) from peracetylatedtrisaccharide 21′ was dissolved in 0.5 ml of anhydrous CH₃CN at roomtemperature. To this solution was added anhydrous LiBr (45 mg, 0.52mmol). The mixture was stirred for 3 hours. After aqueous work-up, thesolvent was evaporated and the residue was separated by chromatographyon silica gel to give compound 25′ (91 mg, 90%). 25′: ¹H NMR (300MHz,CDCl₃) δ6.04 (d, J=3.6 Hz, 1 H, anomeric H).

EXAMPLE 39 Preparation of Trisaccharide Donor 26′

The trisaccharide donor 25′ (91 mg, 0.093 mmol) was dissolved in 2 ml ofanhydrous THF at 0° C. To this solution was added LiSPh (100 ml, 0.103mmol). The reaction was run at 0° C. for half hour. The solvent wasremoved and the residue was separated by chromatography on silica gel togive compound 26′ (61 mg, 66%).

26′: IR (film) 3000, 2100, 1750, 1680, 1500 cm⁻¹; ¹H NMR (300MHz, CDCl₃)δ7.61 (m, 2H) 7.39 (m, 3H), 5.50 (d, J=9.1 Hz, 1H), 5.35 (m, 2H), 5.11(m, 2H), 4.96 (dt, J=10.5, 3.5 Hz, 1H), 4.84 (dd, J=10.2, 3.0 Hz, 1H),4.50 (m, 4H), 4.16 (m, 3H), 3.59-3.90 (m, 8H), 2.15 (s, 3H), 2.10 (s,3H), 2.08 (s, 3H), 2.06 (s, 6H), 2.05 (s, 3H), 2.04 (s, 3H), 1.97 (s,3H), 1.87 (s, 3H).

EXAMPLE 40 Preparation of Trisaccharide Donor 27′

The trisaccharide 21′ (860 mg, 0.722 mmol) was dissolved in 2 ml ofpyridine and 1 ml of Ac₂O in the presence of 10 mg of DMAP. The reactionwas run at 0° C. to room temperature for overnight. After aqueouswork-up, the solvent was removed and the residue was dissolved in 10 mlof MeOH and 5 ml of EtOAc at room temperature. To this solution wereadded Na₂HPO₄ (410 mg, 2.89 mmol) and 20% Na—Hg (1.0 g, 4.35 mmol). Thereaction was run for 2 hours and aqueous work-up followed. After removalof the organic solvent, the residue was separated by chromatography onsilica gel to give N-acetyl trisaccharide glycal (740 mg, 94%). Thetrisaccharide glycal (624 mg, 0.571 mmol) was dissolved in 3 ml ofanhydrous CH₃CN at −40° C. To the solution were added NaN₃ (56 mg, 0.86mmol) and CAN (939 mg, 1.71 mmol) subsequently. The mixture was stirredat −40° C. for 4 hours. After aqueous work-up, the organic solvent wasremoved and the residue was separated by chromatography on silica gel togive a mixture of α and β azidonitrate anomers (191 mg, 27%). Thismixture of anomers (172 mg, 0.137 mmol) was dissolved in 1 ml of CH₃CNat room temperature. To the solution were added EtN(i-Pr)₂ (24 μl, 0.137mmol) and PhSH (42 μl, 0.410 mmol) subsequently. The reaction wascomplete in half hour and the solvent was blown off. Separation oncolumn afforded desired hemiacetal (170 mg). This hemiacetal wasdissolved in 1 ml of CH₂Cl₂ at room temperature. To the solution wereadded 1 ml of CCl₃CN and 500 mg of K₂CO₃. The reaction was run at roomtemperature for overnight. After filtration through a pad of celite, theorganic solvent was removed and the residue was separated bychromatography on silica gel to give desired α-trichloroacetimidate 27′(70 mg, 42%). 27′: IR (film) 3000, 2120, 1670, 1490, 1450 cm⁻¹; ¹H NMR(300 MHz, CDCl₃) δ8.62 (s, 1H), 7.06-7.48 (m, 30H), 6.44 (d, J=3.0 Hz,1H), 5.21 (d, J=11.4 Hz, 1H), 5.03 (m, 2H), 4.89 (d, J=11.0 Hz, 1H),4.80 (d, J=11.3 Hz, 1H), 4.69 (d, J=11.1 Hz, 1H), 4.64 (d, J=7.8 Hz,1H), 4.44-4.58 (m, 5H), 4.18-4.36 (m, 7H), 3.96-4.08 (m, 3H), 3.72-3.81(m, 3H), 3.38-3.62 (m, 6H), 3.31 (dd, J=7.0, 2.7 Hz, 1H), 1.59 (s, 3H),1.31 (s, 3H), 1.14 (s, 3H); HRMS(FAB) calc. for C₆₈H₇₄O₁₅N₅Cl₃Na [M+Na+]1316.4145, found 1316.4110.

EXAMPLE 41 Coupling of trisaccharide donor 23α′ with methyl N-FmocSerinate

To a solution of trisaccharide donor 23α′ (70 mg, 0.046 mmol), methylN-Fmoc serinate (23.4 mg, 0.068 mmol) and 300 mg of 4 Å molecular sievein 0.5 ml of THF at −78° C. was added TMSOTf (4.6 μl, 0.023 mmol). Thereaction was stirred at −35° C. for overnight. The reaction was quenchedby Et₃N and the solution was filtered through a pad of celite. Thefiltrate was evaporated and the residue was separated by chromatographyon silica gel to give 29α′ (70 mg, 90%) and 29β′ (7.0 mg, 9.0%).

EXAMPLE 42 Coupling of trisaccharide donor 24′ with benzyl N-Fmocserinate

To a solution of trisaccharide donor 24′ (33 mg, 0.030 mmol), benzylN-Fmoc serinate (33.0 mg, 0.075 mmol) and 100 mg of 4 Å molecular sievein 0.3 ml of THF at −78° C. was added TMSOTf (6.0 μl, 0.030 mmol). Thereaction was stirred from −78° C. to room temperature for 2 hours. Thereaction was quenched by Et₃N and the solution was filtered through apad of celite. The filtrate was evaporated and the residue was separatedby chromatography on silica gel to give 30′ (8.6 mg, 22%, α:β 2:1). 30′:IR (film) 3400, 3000, 2100, 1740, 1500 cm⁻¹; ¹H NMR (300MHz, CDCl₃)δ6.25 (d, J=8.4 Hz, 2/3H), 5.90 (d, J=8.6 Hz, 1/3H), 5.76 (d, J=9.0 Hz,1/3H), 5.71 (d, J=9.0 Hz, 2/3); MS(CI) 1306 [M⁺].

EXAMPLE 43 Coupling of trisaccharide donor 25α′ with benzyl N-Fmocserinate

To a solution of benzyl N-Fmoc serinate (45 mg, 0.107 mmol), AgClO₄(37.0 mg, 0.179 mmol) and 200 mg of 4 Å molecular sieve in 0.6 ml ofanhydrous CH₂Cl₂ was added a solution of trisaccharide donor 25α′ (88mg, 0.0893 mmol) in 0.5 ml of CH₂CL₂ slowly. The reaction was run atroom temperature for overnight. After filtration through a pad ofcelite, the solvent was removed and the residue was separated bychromatography on silica gel to give the coupling product 30′ (66 mg,56%, α:β 3.5:1).

EXAMPLE 44 Coupling of trisaccharide donor 26β′ with benzyl N-Fmocserinate

To a solution of benzyl N-Fmoc serinate (45 mg, 0.107 mmol),trisaccharide donor 26β′ (23 mg, 0.023 mmol) and 50 mg of 4 Å molecularsieve in 1.0 ml of anhydrous CH₂Cl₂ at 0 DC was added a solution of NIS(6.2 mg, 0.027 mmol) and TfOH (0.24 μl, 0.003 mmol) in 0.5 ml of CH₂Cl₂slowly. The reaction was run at 0° C. for 1 hour. The reaction wasquenched by Et₃N and aqueous work-up followed. The organic solvent wasdried over Na₂SO₄. After removal of the solvent, the residue wasseparated by chromatography on silica gel to give the coupling product30′ (12.1 mg, 40%, α:β2:1).

EXAMPLE 45 Coupling of trisaccharide donor 27α′ with benzyl N-Fmocserinate

To a solution of trisaccharide donor 27α′ (40.1 mg, 0.029 mmol), benzylN-Fmoc serinate (18.0 mg, 0.044 mmol) and 200 mg of 4 Å molecular sievein 2.0 ml of THF at −20° C. was added TMSOTf (1.8 μl, 0.009 mmol). Thereaction was stirred from −20° C. to room temperature for 3 hours. Thereaction was quenched by Et₃N and aqueous work-up followed. After driedover Na₂SO₄, the filtrate was evaporated and the residue was separatedby chromatography on silica gel to give 31′ (24 mg, 51%). 31′: IR(film)3000, 2920, 2860, 2100, 1720, 1665, 1500, 1480, 1450 cm⁻¹; ¹H NMR (300MHz, CDCl₃) δ7.78 (m, 2H), 7.65 (d, J=7.5 Hz, 1H), 7.60 (d, J=7.5 Hz,1H), 7.20-7.42 (m, 39H), 6.18 (d, J=7.8 Hz, 1H), 6.05 (d, J=7.3 Hz, 1H),5.23 (s, 2H), 4.95-5.02 (m, 3H), 4.80 (s, 2H), 4.78 (d, J=2.8 Hz, 1 H,anomeric H), 4.72 (s, 2H), 4.58 (m, 4H), 4.37-4.52 (m, 6H), 4.24-4.31(m, 2H), 4.20 (m, 1H), 4.08 (m, 2H), 3.92-4.02 (m, 5H), 3.78-3.85 (m,5H), 3.65 (m, 1H), 3.58 (t, J=6.2 Hz, 1H), 3.36-3.46 (m, 5H), 3.26 (dd,J=7.5, 2.8 Hz, 1H), 1.85 (s, 3H), 1.48 (s, 3H), 1.34 (S, 3H); HRMS(FAB)calc. for C₉₀H₉₅O₁₉N₅Na [M+Na+] 1572.6520, found 1572.6550.

EXAMPLE 46

Coupling of trisaccharide donor 28′ with benzyl N-Fmoc serinate:

To a solution of trisaccharide donor 28′ (α:β 1:1)(162 mg, 0.163 mmol),benzyl N-Fmoc serinate (48.0 mg, 0.097 mmol) and 300 mg of 4 Å molecularsieve in 2.0 ml of THF at −78° C. was added BF₃Et₂O (0.5 eq., 0.082mmol) in CH₂Cl₂. The reaction was stirred from −78° C. to roomtemperature for 2 hours. The reaction was quenched by Et₃N and aqueouswork-up followed. After dried over Na₂SO₄, the filtrate was evaporatedand the residue was separated by chromatography on silica gel to give32′ (81 mg, 67%). 32′: IR(film) 3420, 3020, 2940, 2880, 2120, 1745,1500, 1450 cm⁻¹, ¹H NMR (300 MHz, CDCl₃) δ7.74 (d, J=7.4 Hz, 2H), 7.60(t, J=7.5 Hz, 2H), 7.20-7.39 (m, 9H), 5.85 (d, J=8.4 Hz, 1H), 5.48 (d,J=12.6 Hz, 1H), 5.32 (d, J=3.4 Hz, 1H), 5.19 (d, J=12.6 Hz, 1H), 5.07(d, J=8.0 Hz, 1H), 4.90 (dd, J=10.3, 3.4 Hz, 1H), 4,83 (t, J=10.3 Hz,1H), 4.72 (d, J=9.3 Hz, 1H), 4.67 (d, J=9.6 Hz, 1H), 3.80-4.47 (m, 9H),3.62 (t, J=9.5 Hz, 1H), 3.32-3.42 (m, 2H), 2.93 (d, J=7.7 Hz, 1 H), 2.14(s, 3H), 2.08 (s, 6H), 2.04 (s, 3H), 2.02 (s, 3H), 1.95 (s, 3H), 1.55(s, 3H), 1.34 (s, 3H).

EXAMPLE 47 Coupling of trisaccharide donor 28β′ with benzyl N-Fmocserinate

To a solution of trisaccharide donor 28β′ (12.0 mg, 0.012 mmol), benzylN-Fmoc serinate (9.0 mg, 0.022 mmol) and 100 mg of 4 Å molecular sievein 0.5 ml of THF at −40° C. was added BF₃ Et₂O (1.5 eq., 0.018 mmol) inCH₂Cl₂. The reaction was stirred from −40° C. to room temperature for 2hours. The reaction was quenched by Et₃N and aqueous work-up followed.After dried over Na₂SO₄, the filtrate was evaporated and the residue wasseparated by chromatography on silica gel to give 32′ (5.2 mg, 35%).

2.3-ST Antigen Precursor

A mixture of thioethyl glycosyl donor 30 (52 mg, 0.064 mmol) and 6-TBDMSacceptor 31 (94 mg, 0.13 mmol) were azeotroped with benzene (4×50 mL),then placed under high vacuum for 1 h. The mixture was placed undernitrogen, at which time 4 Å mol sieves (0.5 g), CH₂Cl₂ (5 mL), and NIS(36 mg, 0.16 mmol) were added. The mixture was cooled to 0° C., andtrifluoromethanesulfonic acid (1% in CH₂Cl₂, 0.96 mL, 0.064 mmol) wasadded dropwise over 5 min. The suspension was warmed to ambienttemperature immediately following addition and stirred 20 min. Themixture was partitioned between EtOAc (50 mL) and sat. NaHCO₃ (50 mL).The phases were separated, and the organic phase washed with brine (50mL), dried (Na₂SO₄), and concentrated. The residue was purified by flashchromatography on silica gel (4:1, EtOAc:hexanes) to provide 59 mg (62%)of the trisaccharide 32 as a colorless crystalline solid.

Le^(y) Antigen Precursor

To thiodonor 32 (44.0 mg, 29.5 μmol) and acceptor 31 (42.4 mg, 59.0μmol) (azeotroped 3 times with toluene) were added CH₂Cl₂ and freshlyactivated 4 Å molecular sieves. The mixture was stirred for 20 min thencooled to 0° C. N-iodosuccinimide (16.6 mg, 73.8 μmol) was added,followed by the dropwise addition of a 1% solution of TfOH in CH₂Cl₂.The red mixture was stirred at 0° C. for 5 min. then was diluted withEtOAc. The organic phase was washed with sat. NaHCO₃, sat. Na₂S₂O₃, andbrine, dried over MGSO₄, then concentrated in vacuo. Flashchromatography (1:1 EtOAc/CH₂Cl₂ to 2:1 EtOAc/CH₂Cl₂) afforded 43.2 mg(68%) of the coupled product 34.

Coupling of b-Trichloroacetimidate with Protected Threonine

To a solution of trichloroacetimidate 35 (98 mg, 0.13 mmol), threoninederivative 36 (70 mg, 0.16 7 mmol) and 100 mg 4 Å molecular sieve in 6ml of anhydrous CH₂Cl₂ at −30° C. was added TMSOTf (14 mL, 0.07 mmol).The reaction was stirred at −30° C. for 1 hour, then neutralized withEt₃N. The reaction mixture was filtered through a pad of celite andwashed with EtOAc. The filtrate was washed with H₂O, brine and driedover anhydrous Na₂SO₄. After evaporation of the solvent, the residue wasseparated by chromatography on silica gel to give β-product 37β (56 mg,42%) and the α-product 37α (57 mg, 42%).

Discussion

The synthetic approach taken in the present invention encompasses fourphases (FIG. 2). First, the complete glycodomain is assembled in theform of an advanced glycal. This is followed by efficient coupling to aserine, threonine or analogous residue. The third stage involves peptideassembly incorporating the full glycosyl domain amino acids into thepeptide backbone. The concluding phase involves global deprotectioneither in concurrent or segmental modes.

The synthetic starting point was the readily available glycal 2 (FIG.3). (Oxidation of this compound with dimethyldioxirane and subsequentcoupling of the resultant epoxide with 6-O-TIPS-galactal was promoted byZnCl₂ in the standard way. Toyokuni, T.; Singhal, A. K.; Chem. Soc. Rev.1995, 24, 231. Acetylation of the crude product yielded disaccharide 3in high yield and stereoselectivity. Removal of the TIPS protectinggroup under mild conditions set the stage for attachment of sialic acidto acceptor 4. The use of sialyl phosphite 5 as the donor, underpromotion of catalytic amounts of TMSOTf, consistently provided highyields (80-85%) of a 4:1 mixture of products. Martin, T. J., et al.,Glycoconjugate. 1993, 10, 16. Sim, M. M, et al., J. Am. Chem. Soc. 1993,115, 2260. Thus, the advanced glycal 6 (“2,6-ST glycal”) is available infour steps with high efficiency.

The trisaccharide glycal 6 was submitted to azidonitration as shown(FIG. 3). Compound 7 thus obtained in 60% yield lent itself toconversion to a variety of donor constructs (see 8-11). For instance,α-bromide 8 can be used as a donor directly or could be converted toβ-phenylthioglycoside 11 with lithium thiophenoxide in a stereoselectivemanner. Alternatively, mixtures of nitrates 7 was hydrolyzed and theresulting hemiacetal converted to 1:1 mixture of α:βtrichloroacetamidates (9) and diethylphoshites (10) in high yields (FIG.3). (Nitrate hydrolysis: Gauffeny, F., et al., Carbohydr. Chem. 1991,219, 237. Preparation and application of trichloroacetamidates: Schmidt,R. R. and Kinzy, W.; Adv. Carbohydr. Chem. Biochem. 1994, 50, 21.Phosphite donors: Kondo, H., et al.; J. Org. Chem. 1994, 59, 864.)

TABLE 1 Reaction of 11 with N—FMOC—Ser(OH)—OBn. R = H(12) R = CH₃(13)X(11) Catalyst/Promoter α:β(%) αβ(%) —Br (8α) AgClO₄(1.5eq), 2.6:1 (70%)α only (74%) CH₂Cl₂, rt —O(CNH)CCl₃ BF₃OEt₂(0.5eq),  12:1 (65%) α only(63%) (9β) THF, −30 C. —O(CNH)CCl₃ BF₃OEt₂(0.5eq),   4:1 (66%) α only(60%) (9αβ 1:1) THF, −30 C. —OP(OEt)₂ BF₃OEt₂(0.5eq),  30:1 (30%) —(10αβ 1:1) THF, −30 C.

The availability of various donor types (8-11) enabled the investigationof the direct coupling of (2,6)-ST trisaccharide to benzyl ester ofN-Fmoc-protected L-serine and L-threonine. The results are summarized inTable 1. As with Fmoc protected L-threonine as the acceptor, all of thedonors afforded the α-O glycosyl threonine system in highstereoselectivity. By contrast, the outcome of the coupling reactionswith similarly protected L-serine acceptors was dependent on thecharacter of the donor and on the reaction conditions. In all cases, thedesired α-anomer 12 was the major product. (For previous attempts tocouple a trisaccharide donor to serine, in which β-anomers were isolatedas the major products, see: Paulsen, H. et al., Liebigs Ann. Chem. 1988,75; Iijima, H.; Ogawa, T., Carbohydr. Res. 1989, 186, 95.) With donor 10the ratio of desired α-product:undesired β-glycoside was ca 30:1.

The glycopeptide assembly phase was entered with building units 14 and15, thereby reducing the number of required chemical operations to beperformed on the final glycopeptide. Thus, compounds 14 and 15 wereobtained in two steps from 12 and 13, respectively. The azidefunctionality was transformed directly to N-acetyl groups by the actionof CH₃COSH in 78-80% yield and the benzyl ester was removedquantitatively by hydrogenolysis (FIG. 4). Paulsen, H., et al., LiebigsAnn. Chem. 1994, 381.

The glycopeptide backbone was built in the C-N-terminus direction (FIG.4). Iteration of the coupling step between the N-terminus of a peptideand protected glycosyl amino acid, followed by removal of the FMOCprotecting group provided protected pentapeptide 16. The peptidecoupling steps of block structures such as 12 and 13 proceeded inexcellent yields. Both IIDQ and DICD coupling reagents work well(85-90%). FMOC deprotection was achieved under mild treatment with KF inDMF in the presence of 18-crown-6. Jiang, J., et al., Synth. Commun.1994, 24, 187. The binal deblocking of glycopeptide 16 was accomplishedin three stages: (i) Fmoc removal with KF and protection of the aminoterminus with acetyl group; (ii) hydrogenolysis of the benzyl ester; and(iii) final saponification of three methyl esters, cyclic carbonates andacetyl protection with aqueous NaOH leading to glycopeptide mucin model1 (FIG. 4).

The orthogonal exposure of both N- and C-termini provided an opportunityfor further extension of the glycopeptide constructs via fragmentjoining. In order to demonstrate the viability of such claims, anonapeptide with ST triad 19 was made by means of coupling tripeptide 18to hexapeptide 17 (see FIG. 5). The previous deprotection protocolprovided nonapeptide mucin model 20, wherein the o-glycosylatedserine-threonine triad had been incorporated in the middle of thepeptide.

Vaccination with Tn Cluster Constructs in Mice

The present invention provides anti-tumor vaccines wherein theglycopeptide antigen disclosed herein is attached to the lipopeptidecarrier PamCys. The conjugation of the antigen to the new carrierrepresents a major simplification in comparison to traditional proteincarriers. Tables 2 and 3 compare the immunogenicity of the newconstructs with the protein carrier vaccines in mice. These novelconstructs proved immunogenic in mice. As shown in the Tables, theTn-PamCys constructs elicit high titers of both IgM and IgG after thethird vaccination of mice. Even higher titers are induced after thefifth vaccination. The Tn-KLH vaccine yields stronger overall response.However, the relative ratio of IgM/IgG differs between the two vaccines.Tn-KLH gives higher IgM/IgG ratio than the Tn Pamcys. In a relativesense, the novel Tn-PamCys vaccine elicits a stronger IgG response. Incontrast to protein carrier vaccines, the adjuvant QS-21 does notprovide any additional enhancement of immunogenicity. Accordingly, thePamCys lipopeptide carrier may be considered as a “built-in”immunostimulant/adjuvant. Furthermore, it should be noted that QS-21enhances the IgM response to Tn-PamCys at the expense of IgG titers. Avaccine based on PamCys carriers is targeted against prostate tumors.

TABLE 2 Antibody Titers by Elisa against Tn-Cluster: 10 μg Tncluster-Pam Pre-serum 10 days post 3rd Group IgM IgG IgM IgG 1.1 50 0450 450 1.2 50 0 1350 50 1.3 50 0 4050 150 1.4 0 0 4050 150 1.5 0 0 4501350 10 μg Tn cluster-pam + QS-21 2.1 50 0 1250 50 2.2 0 0 1350 0 2.3 00 1350 50 2.4 0 0 1350 150 2.5 50 0 1350 150 3 μg Tn cluster KLH + QA-213.1 0 0 12150 450 3.2 0 0 12150 4050 3.3 0 0 36450 450 3.4 0 0 36450 4503.5 0 0 36450 1350 3 μg Tn cluster BSA + QS-21 4.1 0 0 450 1350 4.2 0 0150 4050 4.3 0 50 450 450 4.4 0 0 450 150 4.5 0 0 1350 150 0.3 μg/wellantigen plated in alcohol; serum drawn 11 days post 3rd vaccine.

TABLE 3 Antibody Titers by Elisa against Tn-Cluster: Tn Cluster-PamPre-serum (before Post Serum (10 days 5th Vaccination) after 5thVaccination) Group IgM IgG IgM IgG 1.1 2560 200 640 5120 1.2 25.600 8001280 320 1.3 640 160 640 1280 1.4 2560 1280 25.600 5120 1.5 640 51202560 5120 Tn Cluster-Pam + QS-21 2.1 6400 1280 128.000 0 2.2 3200 1605120 200 2.3 3200 1280 16.000 640 2.4 6400 640 8000 200 2.5 5120 8064.000 2560 Tn Cluster-KLH 3.1 6400 1600 25.600 25.600 3.2 2560 3200128.000 25.600 3.3 16.000 8000 128.000 25.600 3.4 640 12.800 5120 25.6003.5 5120 12.800 25.600 3200 Tn-Cluster-BSA 4.1 2560 12.800 2560 * 4.2800 200 128.000 400 4.3 400 2560 6400 400 4.4 800 2560 12800 2560 4.51280 200 3200 3200 0.2 μg/well plated in ethanol. *ND

TABLE 4 Tn-Cluster FACS Anaiysis; Serum Tested 11 Days Post 3rdVaccination. FACS analysis using LSC cell line (Colon Cancer Cell line).Group IgG (% Gated) IgM (% Gated) Tn Cluster Pam 1-1 93.95 16.59 1-219.00 66.15 1-3 54.45 40.51 1-4 46.99 39.98 1-5 3.07 32.83 TnCluster-Pam + QS-21 2-1 12.00 76.78 2-2 2.48 36.76 2-3 20.27 46.41 2-410.64 55.29 2-5 3.37 38.95 Tn-Cluster-KLH 3-1 96.36 66.72 3-2 93.1245.50 3-3 97.55 32.96 3-4 94.72 49.54 3-5 83.93 64.33 Tn-Cluster-BSA 4-180.65 41.43 4-2 90.07 31.68 4-3 42.86 54.03 4-4 95.70 63.76 4-5 92.1451.89

TABLE 5 Results of Tn-trimer-Cys-KLH and Tn-trimer-Cys-BSA (MBScross-linked) Conjugates Amt of Carbohydrate & KLH used for Final Amt ofCarbohydrate % μg of Conjugation Conjugation Recovered Recoveredcarbohydrate/ μg of Conjugate Carbo. KLH Volume Carbohydrate KLHCarbohydrate KLH 100 μl KLH/100 μl Tn-trimer-Cys-KLH 2.0 mg 5.0 mg 4.25ml 141.174 μg 3612.5 μg 7% 72.25% 3.321 85 2.5* 5.65 (3 μg/mouse; 300μl/vial¶) Tn-trimer-Cys-BSA 2.0 2.0 3.25 108.9 2762.5 5.445 100 3.35 851* 10.89 (3 μg/mouse; 170 μl/vial¶) *After concentration. ¶ Approximateamount.

A Total Synthesis of the Mucin Related F1α Antigen

The present invention provides derived mimics of surfaces of tumortissues, based mainly on the mucin family of glycoproteins. Ragupathi,G., et al., Angew. Chem. lnt. Ed. Engl. 1997, 36, 125. (For a review ofthis area see Toyokuni, T.; Singhal, A. K. Chem. Soc. Rev. 1995, 24,231; Dwek, R. A. Chem. Rev. 1996, 96, 683.) Due to their high expressionon epithelial cell surfaces and the high content of clustered 0-linkedcarbohydrates, mucins constitute important targets for antitumorimmunological studies. Mucins on epithelial tumors often carry aberrantα-O-linked carbohydrates. Finn, O. J., et al., Immunol. Rev. 1995, 145,61; Saitoh, O. et al., Cancer Res. 1991, 51, 2854; Carlstedt, I.;Davies, J. R. Biochem. Soc. Trans. 1997, 25, 214. The identified F1αantigens 1′ and 2′ represent examples of aberrant carbohydrate epitopesfound on mucins associated with gastric adenocarcinomas (FIG. 22A).Yamashita, Y., et al., J. Nat. Cancer Inst. 1995, 87, 441; Yamashita,Y., et al., Int. J. Cancer 1994, 58, 349. Accordingly, the presentinvention provides a method of constructing the F1α epitope throughsynthesis. A previous synthesis of F1α is by Qui, D.; Koganty, R. R.Tetrahedron Lett. 1997, 38, 45. Other prior approaches to α-O-linkedglycopeptides include Nakahara, Y., et al., in SyntheticOligasaccharides, Indispensable Probes for the Life Sciences ACS Symp.Ser. 560, pp 249-266 (1994); Garg, H. G., et al., Adv. Carb. Chem.Biochem. 1994, 50, 277; Paulsen, H., et al., J. Chem. Soc., PerkinTrans. 1, 1997, 281; Liebe, B.; Kunz, H. Angew. Chem. lnt. Ed. Engl.1997, 36, 618; Elofsson, M., et al., Tetrahedron 1997, 53, 369;Meinjohanns, E., et al., J. Chem. Soc., Perkin Trans. 1, 1996, 985;Wang, Z.-G., et al., Carbohydr. Res. 1996, 295, 25; Szabo, L., et al.,Carbohydr. Res. 1995, 274, 11.

The F1α structure could be constructed from the three principal buildingunits I−III (FIG. 22A). Such a general plan permits two alternativemodes of implementation. (For a comprehensive overview of glycalassembly, see: Bilodeau, M. T.; Danishefsky, S. J. Angew. Chem. Int. Ed.Engl. 1996, 35, 1381. For applications toward the synthesis ofcarbohydrate tumor antigen based vaccines, see Sames, D., et al., Nature1997, 389, 587; Park, T. K., et al., J. Am. Chem. Soc. 1996, 118, 11488;and Deshpande, P. P.; Danishefsky, S. J. Nature 1997, 387, 164.) First,a GalNAc-serine/threonine construct might be assembled in the initialphase. This would be followed by the extension at the “non-reducing end”(II+III, then I). Alternatively, the entire glycodomain could beassembled first in a form of trisaccharide glycal (I+II). This stepwould be followed by coupling of the resultant trisaccharide donor to aserine or threonine amino acid residue (cf. II). Both strategies aredisclosed herein.

The first synthetic approach commenced with preparation ofmonosaccharide donors 5a′/b′ and 6a′/b′ (FIG. 22B). The protectinggroups of galactal (cf. II) were carefully chosen to fulfill severalrequirements. They must be stable to reagents and conditions in theazidonitration protocol (vide infra). Also, the protecting functionsmust not undermine the coupling step leading to the glycosyl amino acid.After some initial experimentation, galactal 3′ became the startingmaterial of choice. The azidonitration protocol (NaN₃, CAN CH₃CN, −20°C.) provided a 40% yield of 1:1 mixture of 4a′ and 4b′. Lemieux, R. U.;Ratcliffe, R. M. Can. J. Chem. 1979, 57, 1244. Both anomers werehydrolyzed and then converted to a 1:5 mixture of trichloroacetimidates5a′ and 5b′ in good yield (84%). Schmidt, R. R.; Kinzy, W. Adv.Carbohydr. Chem. Biochem. 1994, 50, 84. Alternatively, hydrolysis ofnitrate 4′ followed by use of the DAST reagent (Rosenbrook, Jr. W., etal., Tetrahedron Lett. 1985, 26, 3; Posner, G. H.; Haines, S. R.Tetrahedron Lett. 1985, 26, 5) yielded a 1:1 mixture of fluoride donors6a′ and 6b′. In both cases the α/β anomers were separable, thus allowingthe subsequent investigation of their behavior in the coupling event.The best results obtained from the coupling of donors 5′-6′ to serine orthreonine acceptors bearing the free side chain alcohol, with protectedcarboxy and amino moieties are summarized in Table 5a.

The trichloroacetimidate donor type 5′ provided excellent yields incoupling reactions with the serine derived alcohol 7′. Afteroptimization, donor 5b′ in the presence of TMSOTf in THF (entry 2, Table5a) provided 86% yield of pure α-product 9′. Interestingly, the donor5a′ also provided α-glycoside 9′ exclusively. The coupling of donor 5b′to threonine, though stereoselective, was low yielding. In this instancethe fluoride donors 6a′ and 6b′, promoted by Cp₂ZrCl₂/AgClO₄ provideddesired glycosyl threonine 10′ in excellent yield (82-87%) though withsomewhat reduced selectivity (6:1, α:β). Ogawa, T. Carbohydrate Res.1996, 295, 25. Thus, both sets of donors proved complementary to oneanother and glycosyl serine 9′ as well as glycosyl threonine 10′ were inhand in high yield and with excellent margins of stereoselectivity. Itwas found that the configurations at the anomeric centers of thesedonors had no practical effect on the stereochemical outcome of theircoupling steps. This result differs from the finding with commonly used2-deoxy-2-azido-tri-O-acetylgalactose-1-O-trichloroacetimidate. SeeSchmidt, R. R.; Kinzy, W., id. In that case each anomer yields adifferent ratio of α/β products (see below).

TABLE 5a R = H(9′) R = CH₃(10′) x Catalyst/promotor α::β(%) α::β(%)—O(CNH)CCl₃(5b′) TMSOTf(0.1eq), 7:3(100%) 7:1(33%) CH₂Cl₂/Hex—O(CNH)CCl₃(5b′) TMSOTf(0.5eq), THF 1:0(86%) 1:0(15%) —O(CNH)CCl₃(5a′)TMSOTf(0.1eq), THF 1:0(66%) — —F (6a′) Cp₂ZrCl₂/AgClO₄ 2:1(89%) 6:1(87%)(2eq), CH₂Cl₂ —F(6b′) Cp₂ZrCl₂/AgClO₄ 2:1(91%) 6:1(82%) (2eq), CH₂Cl₂

The TIPS group at position 6 was quantitatively removed with TBAF andAcOH to give acceptors 11′ and 12′ (FIG. 23). The final coupling tolactosamine donor 13′ was performed in the presence of BF₃OEt₂ in THF.The crude products from this apparently stereoselective coupling stepwere converted to compounds 14′ and 15′, respectively with thiolaceticacid. Paulsen, H., et al., Liebigs Ann.Chem. 1994, 381. These glycosylamino acids represent suitable units for the glycopeptide assembly. Inorder to confirm their structure, we executed global deprotection. Thiswas accomplished in five steps yielding free F1α antigen 1′ and 2′ in70% and 73% yield, respectively (FIG. 23). The glycosidic linkages werenot compromised under the conditions of the acidic and basicdeprotection protocols.

A direct coupling Is provided of trisaccharide donors which aresynthesized through glycal assembly (Bilodeau, M. T.; Danishefsky, S. J.Angew. Chem. Int. Ed. Engl. 1996, 35, 1381) using suitably protectedserine or threonine amino acids. This logic was discussed earlier underthe formalism I+II followed by coupling with III. The trisaccharidedonors 23′-27′ were prepared as outlined in FIG. 24. Readily availablelactal 16′ (Kinzy, W.; Schmidt, R. R. Carbohydrate Res. 1987, 164, 265)was converted to the thio-donor 17′ via a sequence of theiodo-sulfonamidation and subsequent rearrangements with ethanethiol inthe presence of LiHMDS. Park, T. K., et al., J. Amer. Chem. Soc., 1996,118, 11488. The MeOTf-promoted coupling to galactals 18′ and 19′provided the trisaccharide glycals 20′ and 21′ in excellent yield andstereoselectivity. Reductive deprotection of the benzyl groups and thesulfonamide in 20′ and subsequent uniform acetylation of the crudeproduct yielded glycal 22′. The azidonitration of glycal 20′-22′provided intermediate azidonitrates, which were converted to thecorresponding donors 23′-27′.

The results of couplings of these trisaccharide donors with suitableserine/threonine derived acceptors are summarized in Table 6. Theprotection pattern again had a profound effect on the reactivity andstereoselectivity of the coupling. Despite the seemingly large distancebetween the hydroxyl and other functional groups of the lactose domainfrom the anomeric center, these substituents strongly affects thestereochemical outcome. Qualitatively, uniform protection offunctionality with electron donating groups (cf. benzyl) leads to a veryreactive donor by stabilizing the presumed oxonium cation. By contrast,electron withdrawing protecting groups tend to deactivate the donor inthe coupling step. Andrews, C. W., et al., J. Org. Chem. 1996, 61, 5280;Halcomb, R. L.; Danishefsky, S. J. I. Am. Chem. Soc. 1989, 111, 6656.Such deactivation may also confer upon a donor some stereochemicalmemory in terms of sensitivity of coupling to the originalstereochemistry of the donor function at the anomeric center. As shownin Table 6, per-O-benzyl-protected donor 23′ was highly reactive at −78°C. providing product 28′ in 90% yield and high stereoselectivity (10:1,first entry, Table 6). A dramatic difference was seen upon changing theoverall protection from per-O-benzyl to per-O-acetyl groups asdemonstrated in the case of donor 24′. The yield and stereoselectivityof the coupling step were diminished. Comparable results were obtainedwith donors 25′ and 26′.

In the case of compounds 27′ and 28′, where the galactosamine ring wasconformationally restricted by engaging the 3- and 4-positions in thecyclic acetonide, an even more surprising finding was registered. Donor27α′ with a per-O-benzyl protected lactosamine disaccharide affordedonly the desired α-anomer 31′. However, a mixture oftrichloroacetimidates as well as the pure β anomer of 28′ yieldedundesired β anomer 32′ exclusively. Thus, a modification of theprotection pattern at a relatively distant site on the second and thirdcarbohydrate units (from the ring containing the donor function) exerteda profound reversing effect on the stereoselectivity of glycosidation.Conformational limitations imposed on a ring within the donor ensembleby cyclic protecting groups can influence donor reactivity, as judged byrates of hydrolysis. Wilson, B. G.; Fraser-Reid, B. J. Org. Chem. 1995,60, 317; Fraser-Reid, B., et al., J. Am. Chem. Soc,. 1991, 113, 1434.Protecting groups, via their electronic, steric and conformationalinfluences, coupled with salvation effects, can strongly modulate thecharacteristics of glycosyl donors. Thus, longer range effects cannot beaccurately predicted in advance in the glycosidation of serine andthreonine side chain hydroxyls.

TABLE 6 R₁ R₂ R₃ X R₄ Catalyst/Promotor α::β(%) Bn Bn PhSO₂HNO(CNH)CCl₃(23′a) Me TMSOTf(0.5eq), THF  10:1(90%) 29′ Ac Ac AcHNO(CNH)CCl₃(24′α/β 3:1) Bn TMSOTf(1.0eq), THF   2:1(22%) 30′ Ac Ac AcHNBr (25′α) Bn AgClO₄(1.5eq), CH₂Cl₂ 3.5:1(56%) 30′ Ac Ac AcHN SPh (26′β)Bn NIS/TfOH, CH₂Cl₂   2:1(40%) 30′ Me₂C Bn AcHN O(CNH)CCl₃(27′α) BnTMSOTf(0.3eq), THF   1:0(50%) 31′ Me₂C Ac N₃ O(CNH)CCl₃(28′α/β 1:1) BnBF₃Et₂O(0.5eq),THF   0:1(67%) 32′ Me₂C Ac N₃ O(CNH)CCl₃(28′β) BnBF₃Et₂O(1.5eq), THF   0:1(35%) 32′

Accordingly, the present invention demonstrates unexpected advantagesfor the cassette approach wherein prebuilt stereospecificallysynthesized a-O-linked serine or threonine glycosides (e.g., 9′ and 10′)are employed to complete the saccharide assembly.

What is claimed is:
 1. A method of treating cancer in a subjectsuffering therefrom comprising administering to the subject atherapeutically effective amount of a glycoconjugate having thestructure:

wherein the linker is —O—, —NR_(G)—, —NP_(G)(CR_(H)R_(J))_(K)NR_(K)—,NR_(G)(CR_(H)R_(J))_(K)NR_(K)(C═O)(CR_(H)R_(J))_(K)S—,—(CR_(H)R_(J))_(K)NR_(K)—, —O(CR_(H)R_(J))_(K)NR_(K)—, an oligoesterfragment comprising from 2 to about 20 hydroxy acyl residues, a peptidicfragment comprising from 2 to about 20 amino acyl residues, or a linearor branched chain alkyl or aryl carboxylic ester, wherein eachoccurrence of k is independently 1-5; wherein each occurrence of R_(G),R_(H), R_(J) or R_(K) is independently hydrogen, a linear or branched,substituted or unsubstituted, cyclic or acyclic alkyl moiety, or asubstituted or unsubstituted aryl moiety; wherein the crosslinker is amoiety derived from a crosslinking reagent capable of conjugating asurface amine of the carrier with a terminal thiol of the linker;wherein the carrier is a protein or lipid; wherein n is 1, 2, 3 or 4;wherein q is 0 or 1; wherein each occurrence of R_(A), R_(B) and R_(C)is independently H or methyl; and wherein each occurrence of R_(D),R_(E) and R_(F) independently comprises a carbohydrate domain having thestructure:

 wherein a, b, c, d, e, f, g, h, i, x, y and z are each independently 0,1, 2 or 3, with the proviso that R_(D), R_(E), and R_(F) arecarbohydrates independently comprised of fluranose or pyranose moieties,whereby the sum of b and c is 1 or 2, the sum of d and f is 1 or 2, andthe sum of g and i is 1 or 2, and with the proviso that x, y and z arenot simultaneously 0; wherein R₀ is a hydrogen, linear or branched chainalkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R₁, R₂,R₃, R₄, R₅, R₆, R₇, R₈, and R₉ is independently hydrogen, OH, OR^(i),NH₂, NHCOR^(i), F, CH₂OH, CH₂OR^(i), a substituted or unsubstitutedlinear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-,di- or tri)acyloxyalkyl, arylalkyl, or aryl group; wherein eachoccurrence of R^(i) is independently hydrogen, CHO, CO₂R^(ii), asubstituted or unsubstituted linear or branched chain alkyl, acyl,arylalkyl, or aryl group, or a saccharide moiety having the structure:

 wherein Y and Z are independently NH or O; wherein k, 1, r, s, t, u, vand w are each independently 0, 1 or 2, with the proviso that the v andw bracketed structures represent furanose or pyranose moieties and thesum of l and k is 1 or 2, and the sum of s and u is 1 or 2, and with theproviso that v and w are not simultaneously 0; wherein R′₀ is hydrogen,a linear or branched chain alkyl, acyl, arylalkyl or aryl group; whereineach occurrence of R₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ is independentlyhydrogen, OH, OR^(iii), NH₂, NHCOR^(iii), F, CH₂OH, CH₂OR^(iii), or asubstitute or unsubstituted linear or branched chain alkyl, (mono-, di-or tri)hydroxyalkyl, (mono- di- or tri)acyloxyalkyl, arylalkyl or arylgroup; wherein R₁₆ is hydrogen, CO₂H, CO₂R^(ii), CONHR^(ii), asubstituted or unsubstituted linear or branched chain alkyl or arylgroup; wherein each ocurrence of R^(iii) is independently hydrogen, CHO,CO₂R^(iv), or a substituted or unsubstituted linear or branched chainalkyl, acyl, arylalkyl or aryl group; and wherein each occurrence ofR^(ii) and R^(iv) is independently hydrogen, or a substituted orunsubstituted linear or branched chain alkyl, acyl, arylalkyl or arylgroup, with the limitation that each of R_(D), R_(E), and R_(F) comprisea carbohydrate domain, or truncated or elongated version thereof, thatis present on tumor cells, and with the further limitation that (i) whenq is 0, the linker is —NH(CH₂)₂—O—(CH₂)₂NH—, and the carrier is KLH orhuman serum albumin, then R_(D), R_(E) and R_(F) are not simultaneouslySTn, and (ii) when q is 0, the linker is —NH(CH₂)₃(C═O)—, and thecarrier is ovine serum albumin, then R_(D), R_(E) and R_(F) are notsimultaneously Tn.
 2. A method of treating cancer in a subject sufferingtherefrom comprising administering to the subject a therapeuticallyeffective amount of a glycoconjugate having the structure:

wherein m′, n′ and p′ are integers between about 8 and 20; wherein j isan integer between 1 and about 8; wherein R_(V), R_(A), R_(B) and R_(C)are independently hydrogen, substituted or unsubstituted linear orbranched chain lower alkyl or substituted or unsubstituted phenyl;wherein R_(D), R_(E) and R_(F) are independently a carbohydrate domainhaving the structure:

 wherein a, b, c, d, e, f, g, h, i, x, y and z are each independently 0,1, 2 or 3, with the proviso that R_(D), R_(E), and R_(F) arecarbohydrates independently comprised of furanose or pyranose moieties,whereby the sum of b and c is 1 or 2, the sum of d and f is 1 or 2, andthe sum of g and i is 1 or 2, and with the proviso that x, y and z arenot simultaneously 0; wherein R₀ is a hydrogen, linear or branched chainalkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R₁, R₂,R₃, R₄, R₅, R₆, R₇, R₈, and R₁₀ is independently hydrogen, OH, OR^(i),NH₂, NHCOR^(i), F, CH₂OH, CH₂OR^(i), a substituted or unsubstitutedlinear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-,di- or tri)acyloxyalkyl, arylalkyl, or aryl group; wherein eachoccurrence of R^(i) is independently hydrogen, CHO, CO₂R^(ii), asubstituted or unsubstituted linear or branched chain alkyl, acyl,arylalkyl, or aryl group, or a saccharide moiety having the structure:

 wherein Y and Z are independently NH or O; wherein k, l, r, s, t, u, vand w are each independently 0, 1 or 2, with the proviso that the v andw bracketed structures represent furanose or pyranose moieties and thesum of l and k is 1 or 2, and the sum of s and u is 1 or 2, and with theproviso that v and w are not simultaneously 0; wherein R′₀ is hydrogen,a linear or branched chain alkyl, acyl, arylalkyl or aryl group; whereineach occurrence of R₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ is independentlyhydrogen, OH, OR^(iii) ¹, NH₂, NHCOR^(iii), F, CH₂OH, CH₂OR^(iii), or asubstituted or unsubstituted linear or branched chain alkyl, (mono-, di-or tri)hydroxyalkyl, (mono- di- or tri)acyloxyalkyl, arylalkyl or arylgroup; wherein R₁₆ is hydrogen, CO₂H, CO₂R^(ii), CONHR^(ii), asubstituted or unsubstituted linear or branched chain alkyl or arylgroup; wherein each ocurrence of R^(iii) is independently hydrogen, CHO,CO₂R^(iv), or a substituted or unsubstituted linear or branched chainalkyl, acyl, arylalkyl or aryl group; and wherein each occurrence of R′and R^(iv) is independently hydrogen, or a substituted or unsubstitutedlinear or branched chain alkyl, acyl, arylalkyl or aryl group; with thelimitation that each of R_(D), R_(E), and R_(F) comprise a carbohydratedomain, or truncated or elongated version thereof, that is present ontumor cells.
 3. A method of treating cancer in a subject sufferingtherefrom comprising administering to the subject a therapeuticallyeffective amount of a glycoconjugate having the structure:

wherein each occurrence of R_(A), R_(B) and R_(C) is independently H ormethyl; n is 1, 2, 3 or 4; j is 1-8; t′ is 1-8; s′ is 0 or 1, whereinwhen s′=0, the carrier is a lipid, and when s′=1, the carrier is aprotein; each occurrence of R_(D), R_(E) and R_(F) is independently acarbohydrate domain selected from the group consisting of Tn, TF,2,6-STF, 2,6-STn, 3-Le^(y), 6-Le^(y), 3,6-STn, 2,3-ST, a carbohydratehaving the structure:

 a carbohydrate having the structure:

 a carbohydrate having the structure:

 wherein j′, k′ and l′ are each independently 0, 1 or 2; and a Le^(y)hexasaccharide having the structure:

 wherein j′ and k′ are each independently 0, 1 or
 2. 4. A method ofinducing antibodies in a subject, wherein the antibodies are capable ofspecifically binding with human tumor cells, which comprisesadministering to the subject an amount of a glycoconjugate effective toinduce antibodies which glycoconjugate has the structure:

wherein the linker is —O—, —NR_(G)—, —NR_(G)(CR_(H)R_(J))_(k)NR_(K)—,NR_(G)(CR_(H)R_(J))_(k)NR_(K)(C═O)(CR_(H)R_(J))_(k)S—,—(CR_(H)R_(J))_(k)NR_(K)—, —O(CR_(H)R_(J))_(k)NR_(K)—, an oligoesterfragment comprising from 2 to about 20 hydroxy acyl residues, a peptidicfragment comprising from 2 to about 20 amino acyl residues, or a linearor branched chain alkyl or aryl carboxylic ester, wherein eachoccurrence of k is independently 1-5; wherein each occurrence of R_(G),R_(H), R_(J) or R_(K) is independently hydrogen, a linear or branched,substituted or unsubstituted, cyclic or acyclic alkyl moiety, or asubstituted or unsubstituted aryl moiety; wherein the crosslinker is amoiety derived from a crosslinking reagent capable of conjugating asurface amine of the carrier with a terminal thiol of the linker;wherein the carrier is a protein or lipid; wherein n is 1, 2, 3 or 4;wherein q is 0 or 1; wherein each occurrence of R_(A), R_(B) and R_(C)is independently H or methyl; and wherein each occurrence of R_(D),R_(E) and R_(F) independently comprises a carbohydrate domain having thestructure:

wherein a, b, c, d, e, f, g, h, i, x, y and z are each independently 0,1, 2 or 3, with the proviso that R_(D), R_(E), and R_(F) arecarbohydrates independently comprised of furanose or pyranose moieties,whereby the sum of b and c is 1 or 2, the sum of d and f is 1 or 2, andthe sum of g and i is 1 or 2, and with the proviso that x, y and z arenot simultaneously 0; wherein R₀ is a hydrogen, linear or branched chainalkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R₁, R₂,R₃, R₄, R₅, R₆, R₇, R₈, and R₉ is independently hydrogen, OH, OR^(i),NH₂, NHCOR^(i), F, CH₂OH, CH₂OR^(i), a substituted or unsubstitutedlinear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-,di- or tri)acyloxyalkyl, arylalkyl, or aryl group; wherein eachoccurrence of R¹ is independently hydrogen, CHO, CO₂R^(ii), asubstituted or unsubstituted linear or branched chain alkyl, acyl,arylalkyl, or aryl group, or a saccharide moiety having the structure:

 wherein Y and Z are independently NH or O; wherein k, l, r, s, t, u, vand w are each independently 0, 1 or 2, with the proviso that the v andw bracketed structures represent furanose or pyranose moieties and thesum of l and k is 1 or 2, and the sum of s and u is 1 or 2, and with theproviso that v and w are not simultaneously 0; wherein R′₀ is hydrogen,a linear or branched chain alkyl, acyl, arylalkyl or aryl group; whereineach occurrence of R₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ is independentlyhydrogen, OH, OR^(iii), NH₂, NHCOR^(iii), F, CH₂OH, CH₂OR^(iii), or asubstituted or unsubstituted linear or branched chain alkyl, (mono-, di-or tri)hydroxyalkyl, (mono- di- or tri- )acyloxyalkyl, arylalkyl or arylgroup; wherein R₁₆ is hydrogen, CO₂H, CO₂R^(ii), CONHR^(ii), asubstituted or unsubstituted linear or branched chain alkyl or arylgroup; wherein each ocurrence of R^(iii) is independently hydrogen, CHO,CO₂R^(iv), or a substituted or unsubstituted linear or branched chainalkyl, acyl, arylalkyl or aryl group; and wherein each occurrence ofR^(iii)and R^(iv) is independently hydrogen, or a substituted orunsubstituted linear or branched chain alkyl, acyl, arylalkyl or arylgroup, with the limitation that each of R_(D), R_(E), and R_(F) comprisea carbohydrate domain, or truncated or elongated version thereof, thatis present on tumor cells, and with the further limitation that (i) whenq is 0, the linker is —NH(CH₂)₂—O—(CH₂)₂NH—, and the carrier is KLH orhuman serum albumin, then R_(D), R_(E)and R_(F) are not simultaneouslySTn, and (ii) when q is 0, the linker is —NH(CH₂)₃(C═O)—, and thecarrier is ovine serum albumin, then R_(D), R_(E)and R_(F) are notsimultaneously Tn.
 5. A method of inducing antibodies in a subject,wherein the antibodies are capable of specifically binding with humantumor cells, which comprises administering to the subject an amount of aglycoconjugate effective to induce the antibodies, which glycoconjugatehas the structure:

wherein m′, n′ and p′ are integers between about 8 and 20; wherein j isan integer between 1 and about 8; wherein R_(V), R_(A), R_(B) and R_(C)are independently hydrogen, substituted or unsubstituted linear orbranched chain lower alkyl or substituted or unsubstituted phenyl;wherein R_(D), R_(E) and R_(F) are independently a carbohydrate domainhaving the structure:

 wherein a, b, c, d, e, f, g, h, i, x, y and z are each independently 0,1, 2 or 3, with the proviso that R_(D), R_(E), and R_(F) arecarbohydrates independently comprised of furanose or pyranose moieties,whereby the sum of b and c is 1 or 2, the sum of d and f is 1 or 2, andthe sum of g and i is 1 or 2, and with the proviso that x, y and z arenot simultaneously 0; wherein R₀ is a hydrogen, linear or branched chainalkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R₁, R₂,R₃, R₄, R₅, R₆, R₇, R₈, and R₉ is independently hydrogen, OH, OR_(E),NH₂, NHCOR^(i), F, CH₂OH, CH₂OR^(i), a substituted or unsubstitutedlinear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-,di- or tri)acyloxyalkyl, arylalkyl, or aryl group; wherein eachoccurrence of R^(i) is independently hydrogen, CHO, CO₂R^(ii) asubstituted or unsubstituted linear or branched chain alkyl, acyl,arylalkyl, or aryl group, or a saccharide moiety having the structure:

 wherein Y and Z are independently NH or O; wherein k, l, r, s, t, u, vand w are each independently 0, 1 or 2, with the proviso that the v andw bracketed structures represent furanose or pyranose moieties and thesum of l and k is 1 or 2, and the sum of s and u is 1 or 2, and with theproviso that v and w are not simultaneously 0; wherein R′₀ is hydrogen,a linear or branched chain alkyl, acyl, arylalkyl or aryl group; whereineach occurrence of R₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ is independentlyhydrogen, OH, OR^(iii), NH₂, NHCOR^(iii), F, CH₂OH, CH₂OR^(iii), or asubstituted or unsubstituted linear or branched chain alkyl, (mono-, di-or tri)hydroxyalkyl, (mono- di- or tri-)acyloxyalkyl, arylalkyl or arylgroup; wherein R₁₆ is hydrogen, CO₂H, CO₂R^(ii), CONHR^(ii), asubstituted or unsubstituted linear or branched chain alkyl or arylgroup; wherein each ocurrence of R^(iii) is independently hydrogen, CHO,CO₂R^(iii), or a substituted or unsubstituted linear or branched chainalkyl, acyl, arylalkyl or aryl group; and wherein each occurrence ofR^(ii) and R^(iv) is independently hydrogen, or a substituted orunsubstituted linear or branched chain alkyl, acyl, arylalkyl or arylgroup; with the limitation that each of R_(D), R_(E), and R_(F) comprisea carbohydrate domain, or truncated or elongated version thereof, thatis present on tumor cells.
 6. A method of inducing antibodies in asubject, wherein the antibodies are capable of specifically binding withhuman tumor cells, which comprises administering to the subject anamount of a glycoconjugate effective to induce antibodies whichglycoconjugate has the structure:

wherein each occurrence of R_(A), R_(B) and R_(C) is independently H ormethyl; n is 1, 2, 3 or 4; j is 1-8; t′ is 1-8; S is 0 or 1, whereinwhen s′=0, the carrier is a lipid, and when s′=1, the carrier is aprotein; each occurrence of R_(D), R_(E) and R_(F) is independently acarbohydrate domain selected from the group consisting of Tn, TF,2,6-STF, 2,6-STn, 3-Le^(y), 6-Le^(y), 3,6-STn, 2,3-ST, a carbohydratehaving the structure:

 a carbohydrate having the structure:

 a carbohydrate having the structure:

 wherein j, k′ and l′ are each independently 0, 1 or 2; and a Le^(y)hexasaccharide having the structure:

 wherein j′ and k′ are each independently 0, 1 or
 2. 7. A method ofpreventing recurrence of epithelial cancer in a subject which comprisesadministering an amount of a glycoconjugate effective to induceantibodies against an epithelial cancer cell antigen, whichglycoconjugate has the structure:

wherein the linker is —O—, —NR_(G)—, —NR_(G)(CR_(H)R_(J))_(k)NR_(K)—,NR_(C)(CR_(H)R_(J))_(k)NR_(K)(C═O)(CR_(H)R_(J))_(k)S—,—(CR_(H)R_(J))_(k)NR_(K)—, —O(CR_(H)R_(J))_(k)NR_(K)—, an oligoesterfragment comprising from 2 to about 20 hydroxy acyl residues, a peptidicfragment comprising from 2 to about 20 amino acyl residues, or a linearor branched chain alkyl or aryl carboxylic ester, wherein eachoccurrence of k is independently 1-5; wherein each occurrence of R_(G),R_(H), R_(J) or R_(K)is independently hydrogen, a linear or branched,substituted or unsubstituted, cyclic or acyclic alkyl moiety, or asubstituted or unsubstituted aryl moiety; wherein the crosslinker is amoiety derived from a crosslinking reagent capable of conjugating asurface amine of the carrier with a terminal thiol of the linker;wherein the carrier is a protein or lipid; wherein n is 1, 2, 3 or 4;wherein q is 0 or 1; wherein each occurrence of R_(A), R_(B) and R_(C)is independently H or methyl; and wherein each occurrence of R_(D),R_(E) and R_(F) independently comprises a carbohydrate domain having thestructure:

 wherein a, b, c, d, e, f, g, h, i, x, y and z are each independently 0,1, 2 or 3, with the proviso that R_(D), R_(E), and R_(F) arecarbohydrates independently comprised of furanose or pyranose moieties,whereby the sum of b and c is 1 or 2, the sum of d and f is 1 or 2, andthe sum of g and i is 1 or
 2. and with the proviso that x, y and z arenot simultaneously 0; wherein R₀ is a hydrogen, linear or branched chainalkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R₁, R₂,R₃, R₄, R₅, R₆, R₇, R₈, and R₉ is independently hydrogen, OH, OR^(i),NH₂, NHCOR^(i), F, CH₂OH, CH₂OR^(i), a substituted or unsubstitutedlinear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-,di- or tri)acyloxyalkyl, arylalkyl, or aryl group; wherein eachoccurrence of R′ is independently hydrogen, CHO, CO₂R^(ii), asubstituted or unsubstituted linear or branched chain alkyl, acyl,arylalkyl, or aryl group, or a saccharide moiety having the structure:

 wherein Y and Z are independently NH or O; wherein k, l, r, s, t, u, vand w are each independently 0, 1 or 2, with the proviso that the v andw bracketed structures represent furanose or pyranose moieties and thesum of l and k is 1 or 2, and the sum of s and u is 1 or 2, and with theproviso that v and w are not simultaneously 0; wherein R′₀ is hydrogen,a linear or branched chain alkyl, acyl, arylalkyl or aryl group; whereineach occurrence of R₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ is independentlyhydrogen, OH, OR^(iii), NH₂, NHCOR^(iii), F, CH₂OH, CH₂OR^(iii), or asubstituted or unsubstituted linear or branched chain alkyl, (mono-, di-or tri)hydroxyalkyl, (mono- di- or tri)acyloxyalkyl, arylalkyl or arylgroup; wherein R₁₆ is hydrogen, CO₂H, CO₂R^(ii), CONHR^(ii), asubstituted or unsubstituted linear or branched chain alkyl or arylgroup; wherein each ocurrence of R^(iii) is independently hydrogen, CHO,CO₂R^(iv), or a substituted or unsubstituted linear or branched chainalkyl, acyl, arylalkyl or aryl group; and wherein each occurrence ofR^(ii) and R^(iv) is independently hydrogen, or a substituted orunsubstituted linear or branched chain alkyl acyl, arylalkyl or arylgroup, with the, limitation that each of R_(D), R_(E), and R_(F)comprise a carbohydrate domain, or truncated or elongated versionthereof, that is present on tumor cells, and with the further limitationthat (i) when q is 0, the linker is —NH(CH₂)₂—O—(CH₂)₂NH—, and thecarrier is KLH or human serum albumin, then R_(D), R_(E) and R_(F) arenot simultaneously STn, and (ii) when q is 0, the linker is—NH(CH₂)₃(C═O)—, and the carrier is ovine serum albumin, then R_(D),R_(E) and R_(F) are not simultaneously Tn.
 8. A method of preventingrecurrence of epithelial cancer in a subject which comprisesadministering to a subject an amount of a glycoconjugate effective toinduce antibodies, which glycoconjugate has the structure:

wherein m′, n′ and p′ are integers between about 8 and 20; wherein j isan integer between 1 and about 8; wherein R_(V), R_(A), R_(B) and R_(C)are independently hydrogen, substituted or unsubstituted linear orbranched chain lower alkyl or substituted or unsubstituted phenyl;wherein R_(D), R_(E) and R_(F) are independently a carbohydrate domainhaving the structure:

wherein a, b, c, d, e, f, g, h, i, x, y and z are each independently 0,1, 2 or 3, with the proviso that R_(D), R_(E), and R_(F) arecarbohydrates independently comprised of furanose or pyranose moieties,whereby the sum of b and c is 1 or 2, the sum of d and f is 1 or 2, andthe sum of g and i is 1 or 2, and with the proviso that x, y and z arenot simultaneously 0; wherein R₀ is a hydrogen, linear or branched chainalkyl, acyl, arylalkyl or aryl group; wherein each occurrence of RI, R₂,R₃, R4, R₅, R₆, R₇, R₈, and R₉ is independently hydrogen, OH, OR^(i),NH₂, NHCOR^(i), F, CH₂OH, CH₂OR^(i), a substituted or unsubstitutedlinear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-,di- or tri)acyloxyalkyl, arylalkyl, or aryl group; wherein eachoccurrence of R^(i) is independently hydrogen, CHO, CO₂R^(ii), asubstituted or unsubstituted linear or branched chain alkyl, acyl,arylalkyl, or aryl group, or a saccharide moiety having the structure:

 wherein Y and Z are independently NH or O; wherein k, l, r, s, t, u, vand w are each independently 0, 1 or 2, with the proviso that the v andw bracketed structures represent furanose or pyranose moieties and thesum of l and k is 1 or 2, and the sum of s and u is 1 or 2, and with theproviso that v and w are not simultaneously 0; wherein R′₀ is hydrogen,a linear or branched chain alkyl, acyl, arylalkyl or aryl group; whereineach occurrence of R₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ is independentlyhydrogen, OH, OR^(iii), NH₂, NHCOR^(iii), F, CH₂OH, CH₂OR^(iii), or asubstituted or unsubstituted linear or branched chain alkyl, (mono-, di-or tri)hydroxyalkyl, (mono- di- or tri)acyloxyalkyl, arylalkyl or arylgroup; wherein R₁₆ is hydrogen, CO₂H, CO₂R^(iii), CONHR^(ii), asubstituted or unsubstituted linear or branched chain alkyl or arylgroup; wherein each ocurrence of R^(iii) is independently hydrogen, CHO,CO₂R^(iv), or a substituted or unsubstituted linear or branched chainalkyl, acyl, arylalkyl or aryl group; and wherein each occurrence ofR^(ii) and R^(iv) is independently hydrogen, or a substituted orunsubstituted linear or branched chain alkyl, acyl, arylalkyl or arylgroup; with the limitation that each of R_(D), R_(E), and R_(F) comprisea carbohydrate domain, or truncated or elongated version thereof, thatis present on tumor cells.
 9. A method of preventing recurrence ofepithelial cancer in a subject which comprises administering an amountof a glycoconjugate effective to induce antibodies against an epithelialcancer cell antigen, which glycoconjugate has the structure:

wherein each occurrence of R_(A), R_(B) and R_(C) is independently H ormethyl; n is 1 ,2, 3 or 4; j is 1-8; t′ is 1-8; s′is 0 or 1, whereinwhen s′=0, the carrier is a lipid, and when s′=1, the carrier is aprotein; each occurrence of R_(D), R_(E)and R_(F)is independently acarbohydrate domain selected from the group consisting of Tn, TF,2,6-STF, 2,6-STn, 3-Le^(y), 6-Le^(y), 3,6-STn, 2,3-ST, a carbohydratehaving the structure:

 a carbohydrate having the structure:

 a carbohydrate having the structure:

 wherein j′, k′ and l′ are each independently 0, 1 or 2; and a Le^(y)hexasaccharide having the structure:

 wherein j′ and k′ are each independently 0, 1 or
 2. 10. The method ofclaim 1, 4, or 7, wherein n is 1, q is 1, the linker is—NH(CH₂)_(J)NH(C═O)(CH₂)_(t′)S—, and the glycoconjugate has thestructure:

wherein j and t′ are integers between 1land about
 8. 11. The method ofclaim 3, 6 or 9, wherein in the glycoconjugate n is 1, j is 3 and t′is
 1. 12. The method of any one of claims 2, 5, 8 wherein in theglycoconjugate R_(V), R_(A), R_(B) and R_(C) are each independentlymethyl.
 13. The method of any one of claims 2, 5, 8, wherein in theglycoconjugate R_(V), R_(A), R_(B) and R_(C) are each independentlyhydrogen.
 14. The method of any one of claims 2, 5, or 8 wherein in theglycoconjugate the carbohydrate domains are independentlymonosaccharides or disaccharides.
 15. The method of any one of claims 2,5, or 5 wherein in the glycoconjugate x and y are 0; wherein z is 1; andwherein R₃ is NHAc.
 16. The method of any one of claims 2, 5, or 8wherein in the glycoconjugate h is 0; wherein g and i are 1; wherein R₇is OH; wherein R₀ is hydrogen; and wherein R₈ is hydroxymethyl.
 17. Themethod of any one of claims 2, 5, or 8 wherein in the glycoconjugate m′,n′ and p′ are each 14; and j is
 3. 18. The method of any one of claims2, 5, or 8 wherein in the glycoconjugate each amino acyl residue thereinhas an L-configuration.
 19. The method of claim 1, 2, 4, 5, 7, 8, or 10wherein in the glycoconjugate each occurrence of R_(D), R_(E) and R_(F)is independently a carbohydrate domain selected from the groupconsisting of TF, 2,6-STF, 3-Le^(y), 6-Le^(y), 2,3-ST, a carbohydratehaving the structure:

a carbohydrate having the structure:

 a carbohydrate having the structure:

 wherein j′, k′ and l′ are each independently 0, 1 or 2; and a Le^(y)hexasaccharide having the structure:

 wherein j′ and k′ are each independently 0, 1 or
 2. 20. The method ofany one of claims 1, 3, 4, 6, 7 and 9, 10 wherein in the glycoconjugateR_(A), R_(B) and R_(C) are each independently H.
 21. The method of anyone of claims 1, 3, 4, 6, 7, and 9, 10 wherein in the glycoconjugateR_(A), R_(B) and R_(C) are each independently Me.
 22. The method ofclaims 2, 3, 5, 6, 8, 9, or 10 wherein in the glycoconjugate R_(D),R_(E), and R_(F) are each independently Tn.
 23. The method of any one ofclaims 1-10 wherein in the glycoconjugate R_(D), R_(E), and R_(F) areeach independently TF.
 24. The method of any one of claims 1-10 whereinin the glycoconjugate R_(D), R_(E), and R_(F) are each independently2,6-STF.
 25. The method of claims 2, 3, 5, 6, 8, 9 or 10 wherein in theglycoconjugate R_(D), R_(E), and R_(F) are each independently 2,6-STn.26. The method of any one of claims 1-10 wherein in the glycoconjugateR_(D), R_(E), and R_(F) are each independently 3-Le^(y) or 6-Le^(y). 27.The method of claim 2, 3, 5, 6, 8, 9, or 10 wherein in theglycoconjugate R_(D), R_(E), and R_(F) are each independently 3,6-STn.28. The method of any one of claims 1-10 wherein in the glycoconjugateR_(D), R_(E) and R_(F) are each independently 2,3-ST.
 29. The method ofany one of claims 1-10, wherein in the glycoconjugate R_(D), R_(E) andR_(F) are each independently a carbohydrate having the structure:


30. The method of any one of claims 1-10, wherein in the glycoconjugateR_(D), R_(E) and R_(F) are each independently a carbohydrate having thestructure:

wherein j′, k′ and l′ are each independently 0, 1 or
 2. 31. The methodany one of claims 1-10, wherein in the glycoconjugate R_(D), R_(E) andR_(F) are each independently a glycophorine antigen having thestructure:


32. The method of any one of claims 1-10, wherein in the glycoconjugateR_(D), R_(E) and R_(F) are each independently an Le^(y) hexasaccharidehaving the structure:

wherein j′ and k′ are each independently 0, 1 or
 2. 33. The method ofany one of claims 1, 4, 7, or 10, wherein in the glycoconjugate thecrosslinker is a fragment having the structure:

whereby said structure is generated upon conjugation of amaleimidobenzoic acid N-hydroxy succinimide ester with a linker.
 34. Themethod of any one of claims 1, 4, or 7, wherein in the glycoconjugateR_(D), R_(E) and R_(F) are not simultaneously each Tn or STn.
 35. Amethod of treating cancer in a subject suffering therefrom comprisingadministering to the subject a therapeutically effective amount of aglycoconjugate having the structure:

wherein j is 1-8; t′ is 1-8; s′is 0 or 1, wherein when s′=0, the carrieris a lipid and when s′=1, the carrier is a protein; R_(A) is hydrogen ormethyl; and R_(D) is selected from the group consisting of Tn, TF,2,6-STF, 2,6-STn, 3-Le^(y), 6-Le^(y), 3,6-STn, 2,3-ST, a carbohydratehaving the structure:

 a carbohydrate having the structure:

 a carbohydrate having the structure:

 wherein j′, k′ and l′ are each independently 0, 1 or 2; and a Le^(y)hexasaccharide having the structure:

 wherein j′ and k′ are each independently 0, 1 or
 2. 36. A method ofinducing antibodies in a subject, wherein the antibodies are capable ofspecifically binding with human tumor cells, which comprisesadministering to the subject an amount of a glycoconjugate effective toinduce antibodies which glycoconjugate has the structure:

wherein j is 1-8; t′ is 1-8; s′is 0 or 1, wherein when s′0, the carrieris a lipid and when s′=1, the carrier is a protein; R_(A) is hydrogen ormethyl; and R_(D) is selected from the group consisting of Tn, TF,2,6-STF, 2,6-STn, 3-Le^(y), 6-Le^(y), 3,6-STn, 2,3-ST, a carbohydratehaving the structure:

 a carbohydrate having the structure:

 a carbohydrate having the structure:

 wherein j′, k′ and l′ are each independently 0, 1 or 2; and a Le^(y)hexasaccharide having the structure:

 wherein j and k′ are each independently 0, 1 or
 2. 37. The method ofclaim 35 or 36, wherein in the glycoconjugate j is 3 and t′ is
 1. 38.The method of claim 35 or 36, wherein in the glycoconjugate R_(D) is Tn.39. The method of claim 35 or 36, wherein in the glycoconjugate R_(D) isTF.
 40. The method of claim 35 or 36, wherein in the glycoconjugateR_(D) is 2,6-STF.
 41. The method of claim 35 or 36, wherein in theglycoconjugate R_(D) is 2,6-STn.
 42. The method of claim 35 or 36,wherein in the glycoconjugate R_(D) is 3-Le^(y) or 6-Le^(y).
 43. Themethod of claim 35 or 36, wherein in the glycoconjugate R_(D) is3,6-STn.
 44. The method of claim 35 or 36, wherein in the glycoconjugateR_(D) is 2,3-ST.
 45. The method of claim 35 or 36, wherein in theglycoconjugate R_(D), R_(E) and R_(F) are each independently acarbohydrate having the structure:


46. The method of claim 35 or 36, wherein in the glycoconjugate R_(D) isa carbohydrate having the structure:

wherein j′, k′ and l′ are each independently 0, 1 or
 2. 47. The method of claim 35 or 36, wherein in the glycoconjugate R_(D) is a glycophorineantigen having the structure:


48. The method of claim 35 or 36, wherein in the glycoconjugate R_(D) isan Le^(y) hexasaccharide having the structure:

wherein j′ and k′ are each independently 0, 1 or
 2. 49. The method ofclaims 1, 2, 3, or 35, wherein the cancer is a solid tumor.
 50. Themethod of claim 1, 2, 3, or 35, wherein the cancer is an epithelialcancer.
 51. The method of claim 1, 3, 4, 6, 7, 9, 10, 35, or 36, whereinin the glycoconjugate the carrier is a protein, and the protein isbovine serine albumin, polylysine or KLH.
 52. The method of claim 1, 3,4, 6, 7, 9, 10, 35, or 36, wherein in the glycoconjugate the carrier isa lipid, and the lipid is tripalmitoyl-S-glycerylcysteinylserine. 53.The method of any one of claims 1-10 and 35 or 36, wherein the methodfurther comprises co-administering a pharmaceutically suitable carrier.54. The method of any one of claims 1-10 and 35-36, wherein the methodfurther comprises co-administering an immunological adjuvant.
 55. Themethod of claim 54, wherein the adjuvant is bacteria or liposomes. 56.The method of claim 55, wherein the adjuvant is Salmonella minnesotacells, bacille Calmette-Guerin, or QS21.
 57. The method of claim 4, 5,6, or 36, wherein the antibodies induced are those that bind to antigensselected from the group consisting of (2,6)-sialyl T, Le^(a), Le^(b),Le^(x), Le^(y), GM1, SSEA-3, and Globo-H hexasaccharide.
 58. The methodof claim 4, 5, 6, or 36, wherein the subject is in clinical remissionor, where the subject has been treated by surgery, has limitedunresected disease.